TITLE: Cell DIVE™ Platform | Antibody Characterization for Multiplexing
AUTHORS: Liz McDonough, Chrystal Chadwick, Fiona Ginty, Christine Surrette, Anup Sood

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the process of validating antibodies (primary/secondary, direct conjugates, and zenon labelled) as per the Cell DIVE™ technology. It also describes the process for determining any antigen effects from the dye inactivation process.</div></div>

[STEPS]
?. [Decoverslipping]
Fill the plastic slide box with  .
[PBS]
?. [Decoverslipping]
Rest the coverslipped slides on the interior raised edges of slide box such that they are inverted (ie. coverslip down and barcode up).
?. [Decoverslipping]
If your next round is a staining round, prepare your dilutions while you are waiting for the coverslips to come off.
?. [Decoverslipping]
Be patient - within -, coverslips should naturally come off.
?. [Calculating Ab Amount for Ab Dilution Cocktails]
The following section should be used when making cocktails of antibodies for any type of experiment including primary/secondary (P/S), antigen effects (AE), direct conjugates (DC), and revalidation (RV). This includes running 2 different P/S antibodies or conjugates with 2 different dyes on the same slide at the same time. For P/S antibodies, the species (Rabbit IgG, Mouse IgG1, etc) must be different to run on the same slide at the same time.
?. [Calculating Ab Amount for Ab Dilution Cocktails]
Determine the total volume that will be needed for the experiment.
?. [Calculating Ab Amount for Ab Dilution Cocktails]
Determine the stock concentration for the antibodies. This should be written on the barcode or label on the antibody. If there is no concentration written on the vial or the datasheet contact either the vendor and/or chemist who prepared it and ask. Provide the catalog number and lot#, if applicable.
?. [Calculating Ab Amount for Ab Dilution Cocktails]
Once a concentration is obtained for an antibody, determine the final concentration the antibodies or conjugates should be run at.If the antibody/DC is new and commercially available and no final concentration is noted on the vendor datasheet, call the vendor to ask. If no final concentration can be recommended, run the new marker at
?. [Calculating Ab Amount for Ab Dilution Cocktails]
For manual workflow, this is typically (200μL*#of slides)
?. [Calculating Ab Amount for Ab Dilution Cocktails]
Use the following formula to calculate the required amount for each antibody (Ab):stock concentration * X = final concentration Ab1 * total volume needed for experiment stock concentration * X = final concentration Ab2 * total volume needed for experiment Solve for X in each case to determine how many μL of each antibody/DC will be required. Subtract the sum of the Ab volumes together from the total volume to get the volume of Ab diluent (3% BSA in PBS) that needs to be added.
For example: if   and   was used, the sum of Ab volumes= . Subtract this volume from the total volume needed for the experiment to obtain the volume of Ab diluent to add to the Abs.
?. [Calculating Ab Amount for Ab Dilution Cocktails]
If you are using secondary antibodies, determine the volume you will need for the secondary in the same manner described above for calculating for the primary antibody/DC. Secondary antibodies are stained at a final concentration of . Determine which secondaries you need based on the isotypes of the P/S antibodies used.
?. [Calculating Ab Amount for Ab Dilution Cocktails]
Determine if there is a dye preference for each marker (ie. Some markers look better in Cy3 or Cy5). If there is no preference, make the marker that has lower expression in Cy3 if possible.
Check to make sure the dyes are different when making cocktails with DCs. For P/S antibodies, different species as well as different dyes must be used for each antibody. For example, a mouse IgG1 antibody and a rabbit IgG antibody could be pooled; an example of pooled secondary antibodies to use would Cy3-Donkey-anti-mouse and Cy5-Donkey-anti-rabbit.
?. [Ab Characterization]
If you are trying to characterize a new primary-secondary antibody, refer to Study Design & Process Flow (Step 27)
?. [Ab Characterization]
If you are trying to validate a new directly conjugated antibody, proceed to New Direct Conjugate Validation Study Design (Step 72).
?. [Ab Characterization]
If you are trying to re-validate a directly conjugated antibody (new lot/batch), proceed to Revalidating Directly Conjugated Antibodies (Step 86).
?. [Ab Characterization]
If you are trying to validate Zenon labelling, proceed to Zenon Labeling (step 98).
?. [Ab Characterization]
If you are studying antigen effects for a given antibody, proceed to Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation (step 123).
?. [Coverslipping]
Slides are coverslipped to protect the tissue from drying out.
?. [Coverslipping]
Mounting media should be removed from the  fridge and brought to .
4 °C
4 Room temperature
?. [Coverslipping]
Slides should be quickly dipped in ddH2O.
?. [Coverslipping]
Take a rainin pipet tip (200μL-green box) and with a scissors clip the end to make a wide bore.
?. [Coverslipping]
Take up  and add to one end of the slide. For leaching markers, use alternate mounting media (50% glycerol, 4% propyl gallate).
[mounting media (4% propyl gallate, 1% DABCO, 90% glycerol)]
?. [Coverslipping]
Place the end of the coverslip at a 45 degree angle and slowly lower; allowing the mounting media to flow under the coverslip across the slide.
?. [Coverslipping]
Make sure there is not mounting media oozing out of the sides of the coverslip; remove any excess by gently blotting on a Kimwipe.
?. [Coverslipping]
Proceed to Imaging as per the Cell DIVE™ Imaging manual or store the slides at  in light protected environment. (Recommendation: image within 24 hours of staining.)
4 °C
?. [Study Design & Process Flow]
Below is a schematic that provides an overview of the experiment design for antibody characterization for primary-secondary staining. Depending on the slide type (ie, cell pellet vs TMA) and scenario (ie, controls on the slide), a certain experimental design is required. The user should map their scenario to the corresponding study design they must execute.   ABCDEF1ScenarioSlide 1Slide 2Slide 3Slide 42Cell PelletsCell pellet with an absolute positive & negative controlStain with primary/secondary antibodies3Cell pellet with or without an absolute positive & negative control; biomarker is a phospho targetStain with primary/secondary antibodiesPhosphatase treatment, followed by primary/ secondary stain4Cell pellet with or without an absolute positive & negative control; primary antibody has a peptide blockStain with primary/secondary antibodiesPeptide block with primary/secondary stain5Cell pellet with or without an absolute positive & negative control; primary antibody does not have a peptide blockStain with primary/secondary antibodiesStain with isotype control followed by secondary stain6TMATMA with positive and negative control tissue and biomarker is a phospho targetStain with primary/secondary antibodiesPhosphatase treatment, followed by primary/secondary stain7TMA with positive and
negative control tissue;
primary antibody has a
peptide blockStain with primary/secondary antibodiesPeptide block with primary/secondary stain8TMA with positive and negative control tissue; primary antibody does not have peptide blockStain with primary/secondary antibodiesStain with isotype
control followed by
secondary stain9TMA does not exist with both positive and negative control tissue; biomarker is a phospho targetPositive control slide: Stain with primary/secondary antibodiesPositive control slide: Phosphatase treatment, followed by primary/secondary stainNegative control slide: Stain with primary/secondary antibodiesNegative control slide: Phosphatase treatment, followed by primary/secondary stain10TMA does not exist with both positive and negative control tissue; primary antibody has a peptide blockPositive control slide: Stain with primary/secondary antibodiesPositive control slide: Peptide block with primary/secondary stainNegative control slide: Stain with primary/secondary antibodiesNegative control slide: Peptide block with primary/secondary stain11TMA does not exist with both positive and negative control tissue; primary antibody has no peptide blockPositive control slide: Stain with primary/secondary antibodiesPositive control slide: Stain with isotype control followed by secondary stainNegative control slide: Stain with primary/secondary antibodiesNegative control slide: Stain with isotype control followed by secondary stain
ABCDEF1ScenarioSlide 1Slide 2Slide 3Slide 42Cell PelletsCell pellet with an absolute positive & negative controlStain with primary/secondary antibodies3Cell pellet with or without an absolute positive & negative control; biomarker is a phospho targetStain with primary/secondary antibodiesPhosphatase treatment, followed by primary/ secondary stain4Cell pellet with or without an absolute positive & negative control; primary antibody has a peptide blockStain with primary/secondary antibodiesPeptide block with primary/secondary stain5Cell pellet with or without an absolute positive & negative control; primary antibody does not have a peptide blockStain with primary/secondary antibodiesStain with isotype control followed by secondary stain6TMATMA with positive and negative control tissue and biomarker is a phospho targetStain with primary/secondary antibodiesPhosphatase treatment, followed by primary/secondary stain7TMA with positive and
negative control tissue;
primary antibody has a
peptide blockStain with primary/secondary antibodiesPeptide block with primary/secondary stain8TMA with positive and negative control tissue; primary antibody does not have peptide blockStain with primary/secondary antibodiesStain with isotype
control followed by
secondary stain9TMA does not exist with both positive and negative control tissue; biomarker is a phospho targetPositive control slide: Stain with primary/secondary antibodiesPositive control slide: Phosphatase treatment, followed by primary/secondary stainNegative control slide: Stain with primary/secondary antibodiesNegative control slide: Phosphatase treatment, followed by primary/secondary stain10TMA does not exist with both positive and negative control tissue; primary antibody has a peptide blockPositive control slide: Stain with primary/secondary antibodiesPositive control slide: Peptide block with primary/secondary stainNegative control slide: Stain with primary/secondary antibodiesNegative control slide: Peptide block with primary/secondary stain11TMA does not exist with both positive and negative control tissue; primary antibody has no peptide blockPositive control slide: Stain with primary/secondary antibodiesPositive control slide: Stain with isotype control followed by secondary stainNegative control slide: Stain with primary/secondary antibodiesNegative control slide: Stain with isotype control followed by secondary stain
?. [Study Design & Process Flow]
Once the study design has been selected, the user must follow the corresponding process flow for the design. The table below captures the required process for each study design.   ABCD1Primary/Secondary Antibody StainPrimary/Secondary Antibody Stain with Phosphatase PretreatmentPrimary/Secondary Antibody Stain with Immunizing Peptide BlockPrimary/Secondary Antibody Stain with Isotype Controls2Clear SlidesClear SlidesClear SlidesClear Slides3Antigen RetrievalAntigen RetrievalAntigen RetrievalAntigen Retrieval4Phosphatase Pretreatment5Block SlidesBlock SlidesBlock SlidesBlock Slides6Dapi Stain, coverslip, background imageDapi Stain, coverslip, background imageDapi Stain, coverslip, background imageDapi Stain, coverslip, background image7Perform peptide & Primary Antibody Incubation8Stain SlidesStain SlidesStain SlidesStain slides with isotype control alongside9ImageImageImageImage10Qualitative AssessmentQualitative AssessmentQualitative AssessmentQualitative Assessment11Quantitative AssessmentQuantitative AssessmentQuantitative AssessmentQuantitative Assessment
ABCD1Primary/Secondary Antibody StainPrimary/Secondary Antibody Stain with Phosphatase PretreatmentPrimary/Secondary Antibody Stain with Immunizing Peptide BlockPrimary/Secondary Antibody Stain with Isotype Controls2Clear SlidesClear SlidesClear SlidesClear Slides3Antigen RetrievalAntigen RetrievalAntigen RetrievalAntigen Retrieval4Phosphatase Pretreatment5Block SlidesBlock SlidesBlock SlidesBlock Slides6Dapi Stain, coverslip, background imageDapi Stain, coverslip, background imageDapi Stain, coverslip, background imageDapi Stain, coverslip, background image7Perform peptide & Primary Antibody Incubation8Stain SlidesStain SlidesStain SlidesStain slides with isotype control alongside9ImageImageImageImage10Qualitative AssessmentQualitative AssessmentQualitative AssessmentQualitative Assessment11Quantitative AssessmentQuantitative AssessmentQuantitative AssessmentQuantitative Assessment
?. [Study Design & Process Flow]
If your study requires Phosphatase Pretreatment, proceed to Controls—Phosphatase Pretreatment of Tissue (step 51).
?. [Study Design & Process Flow]
If your study requires Immunizing Peptide Block, proceed to Controls—Blocking with Immunizing Peptide (step 60).
?. [Study Design & Process Flow]
If your study requires Isotype Controls, proceed to Controls—Isotype Controls (step 66).
?. [Primary-Secondary Staining]
Retrieve primary antibody from the  walk-in refrigerator or  freezer. Be sure to keep  at all times.
4 °C
-20 °C
on ice
?. [Primary-Secondary Staining]
Spin the stock antibody down at . If stock antibodies are in large or odd shaped vials, transfer them to eppendorf tubes and label appropriately with a LIMS sticker (if possible, remove the LIMS label from the original vial and stick it on the eppendorf tube).
Centrifuge: 13000 34, 1 min
?. [Primary-Secondary Staining]
Make up primary antibody dilutions in the antibody diluent solution (/) using the manufacturer suggested concentration of antibody, with the exceptions noted.
[BSA]
[PBS]
If there is no concentration suggested, then use 5 µg/ mL.If the vendor suggests     incubation). If no concentration or recommended dilution is available, use a 1:100 dilution.
?. [Primary-Secondary Staining]
Spin down dilutions at  and keep dilutions . Put antibodies back to proper storage location after making up dilutions.
Centrifuge: 13000 34, 1 min
on ice
?. [Primary-Secondary Staining]
Slides should be in  (from decoverslipping step); remove from PBS and turn slide on its side and gently tap slide on blue pad/Kimwipe to remove solution. Remove as much of the PBS solution without letting the tissue dry out and without touching the tissue.
[PBS]
?. [Primary-Secondary Staining]
Place slides in humidified chamber. Chamber should contain water in the bottom. Move quickly in adding the primary antibody so that slides do not dry out.
?. [Primary-Secondary Staining]
Add a total of  to the slide; cover with parafilm cut to a size just larger than tissue area. Make sure there are no bubbles under the parafilm. Place lid on humidified chamber.
[antibody dilution]
?. [Primary-Secondary Staining]
Incubate for  at .
20 Room temperature
?. [Primary-Secondary Staining]
Retrieve secondary antibody from ; keep  and then spin down at .
-20 °C
on ice
Centrifuge: 13000 34, 1 min
?. [Primary-Secondary Staining]
Make up secondary antibody dilutions in the antibody diluent solution (/). Secondary antibodies are stained at a final concentration of .
[BSA]
[PBS]
?. [Primary-Secondary Staining]
Spin down dilutions for . Keep  in light protected environment while the primary antibody incubation is taking place.
Centrifuge: 13000 34, 1 min
on ice
?. [Primary-Secondary Staining]
After primary antibody incubation is complete, remove parafilm and place slides in coplin jar or staining dish.
?. [Primary-Secondary Staining]
After final PBS wash, turn slide on its side and gently tap slide on blue pad/Kimwipe to remove remaining PBS. Once again remove as much of the liquid without letting the tissue dry out and without touching the tissue.
?. [Primary-Secondary Staining]
Place slides in humidified chamber. Add a total of  to the slide just as suggested above. Cover with parafilm cut to a size just larger than tissue area. Make sure there are no bubbles under the parafilm. Place lid on humidified chamber.
[secondary antibody dilution]
?. [Primary-Secondary Staining]
Incubate for  at .
-20 Room temperature
?. [Primary-Secondary Staining]
Proceed immediately to Coverslipping.
?. [Primary-Secondary Staining]
Remove parafilm and place slides in coplin jar or staining dish.
?. [Controls—Phosphatase Pretreatment of Tissue]
The phosphatase pretreatment of tissue must occur prior to the slide blocking that occurs in Cell DIVE™ Platform | Slide Clearing and Antigen Retrieval. The user is responsible for following the appropriate process flow given the study design for the antibody characterization.
?. [Controls—Phosphatase Pretreatment of Tissue]
Slides should be coming out of  from antigen retrieval.
[PBS]
?. [Controls—Phosphatase Pretreatment of Tissue]
Check to make sure the oven is at .
37 °C
?. [Controls—Phosphatase Pretreatment of Tissue]
Locate the which should be stored in  freezer. Keep the phosphatase  at all times. The 10X buffer and MnCl2 may be thawed and then kept .
-80 °C
on ice
on ice
?. [Controls—Phosphatase Pretreatment of Tissue]
Make up the following solutions:  ABC1Kit ReagentsNo phosphatase pretreamentPhosphatase pretreament210x buffer20 uL20 uL310x MnCl220 uL20 uL4Lambda0 uL8 uL5ddH20160 uL152 uL6Total Volume200 uL 200 uL
ABC1Kit ReagentsNo phosphatase pretreamentPhosphatase pretreament210x buffer20 uL20 uL310x MnCl220 uL20 uL4Lambda0 uL8 uL5ddH20160 uL152 uL6Total Volume200 uL 200 uL
?. [Controls—Phosphatase Pretreatment of Tissue]
Rinse slides for 3x   with ddH2O.
?. [Controls—Phosphatase Pretreatment of Tissue]
Add entire volume () to slides and incubate at  for  in humidified chamber. Do not cover slides with parafilm.
200 µl
37 °C
?. [Controls—Phosphatase Pretreatment of Tissue]
Wash slides for 2x  each in ddH2O with gentle agitation.
?. [Controls—Phosphatase Pretreatment of Tissue]
Proceed to Slide Blocking in Cell DIVE™ Platform | Slide Clearing and Antigen Retrieval for the blocking procedure.
?. [Controls—Blocking with Immunizing Peptide]
The peptide blocking experiment should be done alongside a primary/secondary antibody staining experiment.
?. [Controls—Blocking with Immunizing Peptide]
Retrieve peptide from  walk-in refrigerator or  freezer and keep .
4 °C
-20 °C
on ice
?. [Controls—Blocking with Immunizing Peptide]
Use the manufacturer’s suggested concentration of peptide (if not indicated on datasheet call vendor); if there is not a concentration recommended then use 100-fold molar excess of peptide to antibody.
If you are unsure in how to calculate this please consult your study coordinator.
?. [Controls—Blocking with Immunizing Peptide]
Use the concentration of primary antibody suggested as per in the Primary - Secondary Antibody Section.
?. [Controls—Blocking with Immunizing Peptide]
Dilute peptide and primary antibody in the antibody diluent buffer (/); mix thoroughly and follow vendor’s recommended incubation period. If there is not one suggested then allow reaction to incubate for  at .
[BSA]
[PBS]
4 Room temperature
?. [Controls—Blocking with Immunizing Peptide]
Add peptide & primary antibody solution to slide and follow remaining steps in Primary - Secondary Antibody starting at .
?. [Controls—Isotype Controls]
The isotype control experiment should be done alongside a primary/secondary antibody staining experiment. The isotype control employed should match the IgG type of the primary antibody.
The same isotype control can be used for antibodies that have the same IgG type if the final concentration used for the antibodies is ≤ 5µg/mL. Any antibody that has a final concentration of > 5µg/mL needs its own isotype control even though it may have the same IgG type as another antibody.
?. [Controls—Isotype Controls]
Double-check the correct isotype control is being used.
?. [Controls—Isotype Controls]
If the isotype is a Mouse IgG1 or Rabbit IgG1, use the Cell Signaling Isotype controls. If it is Mouse IgG2a,2b or Rat, etc use the R&D isotype controls.
?. [Controls—Isotype Controls]
Retrieve isotype control from  or  storage location and keep .
4 °C
-20 °C
on ice
?. [Controls—Isotype Controls]
Use equivalent concentration of isotype control as what is used for the primary antibody. Dilute the isotype control in the antibody diluent buffer (/).
[BSA]
[PBS]
?. [Controls—Isotype Controls]
Proceed to Primary - Secondary Antibody starting at .
?. [New Direct Conjugate Validation Study Design]
Refer to the below table to review the experimental design for validating directly conjugated antibodies.  ABCDEFG1Treatment2Primary/Secondary Antibody stainingSlide 13Directly Conjugated Antibody (D:P ratio1), 5 ug/mLSlide 24Directly Conjugated Antibody (D:P ratio1), 10ug/mLSlide 35Directly Conjugated Antibody (D:P ratio2), 5 ug/mLSlide 46Directly Conjugated Antibody (D:P ratio2), 10ug/mLSlide 5
ABCDEFG1Treatment2Primary/Secondary Antibody stainingSlide 13Directly Conjugated Antibody (D:P ratio1), 5 ug/mLSlide 24Directly Conjugated Antibody (D:P ratio1), 10ug/mLSlide 35Directly Conjugated Antibody (D:P ratio2), 5 ug/mLSlide 46Directly Conjugated Antibody (D:P ratio2), 10ug/mLSlide 5
?. [New Direct Conjugate Validation Study Design]
The slides used for this experiment should be the same type that was used in phase I of the Primary-Secondary Staining.
?. [New Direct Conjugate Validation Study Design]
For the Primary-Secondary Staining slide, use the purified form of the primary antibody if the antibody was purified prior to the conjugation (test at the same concentration used in the P/S experiment).
?. [Staining with Directly Conjugated Antibodies]
Retrieve primary antibody and direct conjugates from the  walk-in refrigerator or  freezer. Be sure to keep  at all times.
4 °C
-20 °C
on ice
?. [Staining with Directly Conjugated Antibodies]
Spin the stock antibody down at .
Centrifuge: 13000 34, 1 min
?. [Staining with Directly Conjugated Antibodies]
Make up necessary primary antibody & direct conjugate antibody dilutions in the antibody diluent buffer (/) using amber tubes for direct conjugates.
[BSA]
[PBS]
The concentration for the unconjugated primary antibody should be equivalent to what was used in the initial phase I characterization experiment. For the direct conjugates use   &  .
In the event that the primary antibody was used at   for the initial characterization; then the direct conjugate should be stained with   and   of the direct conjugate for an extended period of time,  .
?. [Staining with Directly Conjugated Antibodies]
Spin the dilutions at  and keep dilutions . Put antibodies and direct conjugates back to proper storage location after making up dilutions.
Centrifuge: 13000 33, 1 min
on ice
?. [Staining with Directly Conjugated Antibodies]
Slides should be in  (from decoverslipping step); remove from PBS and turn slide on its side and gently tap slide on blue pad/Kimwipe to remove solution. Remove as much of the PBS solution without letting the tissue dry out and without touching the tissue.
[PBS]
?. [Staining with Directly Conjugated Antibodies]
Place slides in humidified chamber. Chamber should contain water in the bottom. Move quickly in adding the primary antibody so that slides do not dry out.
?. [Staining with Directly Conjugated Antibodies]
Add a total of  to the slide; cover with parafilm cut to a size just larger than tissue area. Make sure there are no bubbles under the parafilm. Place lid on humidified chamber.
[antibody dilution]
?. [Staining with Directly Conjugated Antibodies]
Incubate for  at .
-20 Room temperature
?. [Staining with Directly Conjugated Antibodies]
Remove parafilm and place slides in coplin jar or staining dish.
?. [Staining with Directly Conjugated Antibodies]
Proceed immediately to Coverslipping.
?. [Staining with Directly Conjugated Antibodies]
Wash slides 3x  with , using gentle agitation on shaker.
[PBS]
?. [Revalidating Directly Conjugated Antibodies]
Refer to the below table to review the experimental design for re-validating directly conjugated antibodies. A total of four slides are required. 1 slide is stained with the old lot, and the remaining 3 are stained with different concentrations of the new lot.  ABCDE1Treatment2Old Lot of direct conjugateSlide 13New Lot of direct conjugate (5 ug/mL)Slide 24New Lot of direct conjugate (10 ug/mL)Slide 35New Lot of direct conjugate (15 ug/mL)Slide 4
ABCDE1Treatment2Old Lot of direct conjugateSlide 13New Lot of direct conjugate (5 ug/mL)Slide 24New Lot of direct conjugate (10 ug/mL)Slide 35New Lot of direct conjugate (15 ug/mL)Slide 4
The slides used for this experiment must be tissue representative of what will be employed for the multiplexing experiment. The purpose is to determine the optimal concentration of direct conjugate to employ for multiplexing.
?. [Staining with Directly Conjugated Antibodies]
Retrieve direct conjugates from the  refrigerator. Be sure to keep  at all times.
4 °C
on ice
?. [Staining with Directly Conjugated Antibodies]
Spin the direct conjugates down at .
Centrifuge: 13000 34, 1 min
?. [Staining with Directly Conjugated Antibodies]
Make up necessary direct conjugate dilutions in the antibody diluent buffer (/) using amber tubes. The new direct conjugate concentrations should have a range with the working concentration of the old direct conjugate as the mid-point.
[BSA]
[PBS]
For example if the old DC had a working concentration of   then the new DC should be tested at concentrations of  ,   and  .
?. [Staining with Directly Conjugated Antibodies]
Spin the dilutions at  and keep dilutions . Put direct conjugates back to proper storage location after making up dilutions.
Centrifuge: 13000 33, 1 min
on ice
?. [Staining with Directly Conjugated Antibodies]
Slides should be in  (from decoverslipping step); remove from PBS and turn slide on its side and gently tap slide on blue pad/Kimwipe to remove solution. Remove as much of the PBS solution without letting the tissue dry out and without touching the tissue.
[PBS]
?. [Staining with Directly Conjugated Antibodies]
Place slides in humidified chamber. Chamber should contain water in the bottom. Move quickly in adding the DC so that slides do not dry out.
?. [Staining with Directly Conjugated Antibodies]
Add a total of  to the slide; cover with parafilm cut to a size just larger than tissue area. Make sure there are no bubbles under the parafilm. Place lid on humidified chamber.
[antibody dilution]
?. [Staining with Directly Conjugated Antibodies]
Incubate for  at .
-20 Room temperature
?. [Staining with Directly Conjugated Antibodies]
Remove parafilm and place slides in coplin jar or staining dish.
?. [Staining with Directly Conjugated Antibodies]
Wash slides 3x for  with , using gentle agitation on shaker.
[PBS]
?. [Staining with Directly Conjugated Antibodies]
Proceed immediately to Coverslipping.
?. [Zenon Labeling Antibodies]
Zenon labeling provides a fast alternative way of conjugating antibodies. An unlabeled antibody is incubated with fluorophore-labeled Fab fragment and then mixed with a non-specific IgG to neutralize any extra unbound Fab fragments. The initial antibody does not require purification, prior to or after labeling.An initial zenon experiment begins by targettng a 3:1 dye to protein ratio and staining at concentrations of  and .  AB1Zenon KitVendor and Cat. No.2Zenon® Alexa Fluor® 555 Rabbit IgGInvitrogen Z253053Zenon® Alexa Fluor® 555 Mouse IgG1Invitrogen Z250054Zenon® Alexa Fluor® 555 Mouse IgG2bInvitrogen Z252055Zenon® Alexa Fluor® 647 Rabbit IgGInvitrogen Z253086Zenon® Alexa Fluor® 647 Mouse IgG1Invitrogen Z250087Zenon® Alexa Fluor® 647 Mouse IgG2bInvitrogen Z25208
AB1Zenon KitVendor and Cat. No.2Zenon® Alexa Fluor® 555 Rabbit IgGInvitrogen Z253053Zenon® Alexa Fluor® 555 Mouse IgG1Invitrogen Z250054Zenon® Alexa Fluor® 555 Mouse IgG2bInvitrogen Z252055Zenon® Alexa Fluor® 647 Rabbit IgGInvitrogen Z253086Zenon® Alexa Fluor® 647 Mouse IgG1Invitrogen Z250087Zenon® Alexa Fluor® 647 Mouse IgG2bInvitrogen Z25208
Use amber tubes during the workflow.
?. [Validating zenon labeling]
Zenon labeling validation is a 3 slide experiment: slide 1 -  P/S, slides 2 and 3 - titration of zenon complexes. Prepare 3 bullet tubes for the initial zenon labeling validation.
?. [Validating zenon labeling]
The IgG concentration of Ab should be known. If unknown, contact vendor. If no concentration can be supplied, assume 200 µg/mL.
?. [Validating zenon labeling]
Initial zenon labeling should be done using  and  with a relevant IgG (rabbit IgG or mouse IgG1, etc.) labeling reagent when using a final staining volume of .This results in a final concentrations of  and , respectively.
2 µg
[antibody]
200 µl
?. [Validating zenon labeling]
Volume of Ab needed for each initial validation should not be more than .
20 µl
?. [Validating zenon labeling]
For each concentration, test a 3:1 molar ratio of zenon to antibody target to begin.
?. [Validating zenon labeling]
Incubate both tubes for at least  at .
4 Room temperature
?. [Validating zenon labeling]
Add an amount of zenon blocking reagent to each tube equivalent to the amount of labeling reagent used (i.e.  for Tube 2,  for Tube 3).
10 µl
20 µl
?. [Validating zenon labeling]
Incubate for  at .
4 Room temperature
?. [Validating zenon labeling]
Using  at 3:1 ratio (slide 2 of the validation): Take   and add . (Pipet up and down to mix instead of vortexing due to low volumes).
[antibody]
[antibody]
[labeling reagent]
?. [Validating zenon labeling]
Using  at 3:1 ratio (slide 3 of the validation): Take  and add . (Pipet up and down to mix instead of vortexing due to low volumes).
[antibody]
[antibody]
[labeling reagent]
?. [Validating zenon labeling]
Bring the volume of each tube to  using antibody diluent (/) for the manual staining.
200 µl
[BSA]
[PBS]
?. [Validating zenon labeling]
For Tube 1, prepare the P/S antibody diluted to   at the appropriate staining concentration. (Staining concentration as per vendor recommendation or based on previous staining data.)
200 µl
Conjugates must be applied to slides within  .
?. [Validating zenon labeling]
Slides should be in  (from decoverslipping step); remove from PBS and turn slide on its side and gently tap slide on blue pad/Kimwipe to remove solution. Remove as much of the PBS solution without letting the tissue dry out and without touching the tissue.
[PBS]
?. [Validating zenon labeling]
Place slides in humidified chamber. Chamber should contain water in the bottom. Move quickly in adding the primary antibody so that slides do not dry out.
?. [Validating zenon labeling]
Add a total of  to each slide; cover with parafilm cut to a size just larger than tissue area. Make sure there are no bubbles under the parafilm. Place lid on humidified chamber.
[antibody dilution]
?. [Validating zenon labeling]
Incubate for  at .
-20 Room temperature
?. [Validating zenon labeling]
Remove parafilm and place slides in coplin jar or staining dish.
?. [Validating zenon labeling]
Wash slides 3x for  with , using gentle agitation on shaker.
[PBS]
?. [Validating zenon labeling]
Proceed immediately to Coverslipping.
?. [If initial Zenon validation didn’t work]
If the initial zenon validation did not work, consider testing a lower ratio of zenon to antibody target. Target 1.5 -- half the dye to protein targeted initially -- at final concentrations of  and .
?. [If initial Zenon validation didn’t work]
Incubate both tubes for at least  at .
4 Room temperature
?. [If initial Zenon validation didn’t work]
Add an amount of zenon blocking reagent to each tube equivalent to the amount of labeling reagent used.(i.e. for Tube 2,  for Tube 3)
2 µl
4 µl
?. [If initial Zenon validation didn’t work]
Incubate for  at .
4 Room temperature
?. [If initial Zenon validation didn’t work]
Bring the volume of each tube to  using antibody diluent (/) for the manual staining.
200 µl
[BSA]
[PBS]
?. [If initial Zenon validation didn’t work]
Conjugates must be applied to slides within .
?. [If initial Zenon validation didn’t work]
Stain for  at .
4 Room temperature
?. [If initial Zenon validation didn’t work]
Using  when using a final staining volume of : Take  and add . (Pipet up and down to mix instead of vortexing due to low volumes).
[antibody]
200 µl
[antibody]
[labeling reagent]
?. [If initial Zenon validation didn’t work]
Using  when using a final staining volume of : Take  and add . (Pipet up and down to mix instead of vortexing due to low volumes).
[antibody]
200 µl
[antibody]
[labeling reagent]
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
There should be a total of 4 slides prepared for testing the dye inactivation/bleaching effects. Slide 1 will not be treated with the dye inactivation solution, Slide 2 will be treated with 1 round of dye inactivation, Slide 3 will be treated with 5 rounds of dye inactivation, and Slide 4 will be treated with 10 rounds of dye inactivation.  ABCDE1Treatment2No Dye InactivationSlide 131 Round of Dye InactivationSlide 245 Rounds of Dye InactivationSlide 3510 Rounds of Dye InactivationSlide 4
ABCDE1Treatment2No Dye InactivationSlide 131 Round of Dye InactivationSlide 245 Rounds of Dye InactivationSlide 3510 Rounds of Dye InactivationSlide 4
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Slides should be coming out of  from the antigen retrieval.
[PBS]
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Keep slide 1 in  .
[PBS]
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Be sure that the 0.5 M NaHCO3 has had a pH read within 7 days.
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Make fresh dye inactivation solution.
Refer to the working solution for dye inactivation. The hydrogen peroxide is added to the solution immediately before adding to the staining dish. This reaction is time sensitive.
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Within one minute of making the dye inactivation solution, pipet  onto slides 2, 3, and 4 in a humidified chamber containing water. (If there are many slides, use a staining dish containing ).
500 µl
[solution]
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Incubate at  for . Do not place parafilm over the slides.
4 Room temperature
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Remove slide 2 and transfer to . Wash 3x for 5 minutes with gentle agitation on shaker and hold in PBS.
[PBS]
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Perform a quick  wash (less than one minute) for slides 3 and 4 and continue bleaching, making a fresh dye inactivation solution for each round.
[PBS]
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
For slide 3, continue to 5 rounds of dye inactivation and for slide 4 continue to 10 rounds of dye inactivation. Perform a quick  wash between each round.
[PBS]
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Proceed immediately to Coverslipping.
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Acquire background images for the slides as per the Cell DIVE Imaging Manual.
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Decoverslip the slides and stain all 4 slides with the same antibody, as per Primary-Secondary Staining or DC staining, depending on the Ab being tested for dye inactivation.
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
After the final round, wash slides 3x for  with , using gentle agitation on shaker.
[PBS]
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Proceed immediately to Coverslipping.
?. [Overview of Experimental Design for Testing Antigen Effects to Dye Inactivation]
Acquire background images for the slides as per the Cell DIVE Imaging Manual.
?. [Go/No Go Decision: Primary Secondary Antibody staining ]
Open images of all slides in your viewer of choice (FIJI or QUPath, for example).
?. [Go/No Go Decision: Primary Secondary Antibody staining ]
Compare antibody images to corresponding control slides.   ABC1Type Of ComparisonType Of EvaluationNormalized To2Specificity• primary/secondary vs. Isotype controls or phosphatase pretreatment
• three positives and one negative control tissue
• if there is not a negative control use an internal negative controlprimary/secondary3Specificity• Primary/secondary of antibody 1 vs. Primary/secondary of antibody 2 vs. primary/secondary of antibody 3 vs. DABAuto-level each4Difference in intensity• Primary/secondary of antibody 1 vs. Primary/secondary of antibody 2 vs. primary/secondary of antibody 3To brightest specific primary
ABC1Type Of ComparisonType Of EvaluationNormalized To2Specificity• primary/secondary vs. Isotype controls or phosphatase pretreatment
• three positives and one negative control tissue
• if there is not a negative control use an internal negative controlprimary/secondary3Specificity• Primary/secondary of antibody 1 vs. Primary/secondary of antibody 2 vs. primary/secondary of antibody 3 vs. DABAuto-level each4Difference in intensity• Primary/secondary of antibody 1 vs. Primary/secondary of antibody 2 vs. primary/secondary of antibody 3To brightest specific primary
?. [Go/No Go Decision: Primary Secondary Antibody staining ]
Compare antibody staining from all vendors to each other.
?. [Go/No Go Decision: Primary Secondary Antibody staining ]
Compare antibody staining to DAB images, if available.
?. [Go/No Go Decision: Primary Secondary Antibody staining ]
There should be a maximal signal to noise ratio.
?. [Go/No Go Decision: Primary Secondary Antibody staining ]
Select the antibody with the best specificity (localization same as vendor/literature/collaborator suggested) and signal.
?. [Go/No Go Decision: Primary Secondary Antibody staining ]
For troubleshooting on staining, refer to Figure 1: Troubleshooting Primary Antibody Characterization Flow Chart.
?. [Go/No Go Decision: Direct Conjugates]
The direct conjugate staining should have comparable localization to the primary/secondary antibody and DAB images.   ABC1Type Of ComparisonType Of EvaluationNormalized To2Specificityprimary/secondary vs. direct conjugatesAuto-level each3Intensityprimary/secondary vs. direct conjugatesNormalize to primary/secondary4Intensitydirect conjugates vs. direct conjugatesNormalize to brightest most specific direct conjugate
ABC1Type Of ComparisonType Of EvaluationNormalized To2Specificityprimary/secondary vs. direct conjugatesAuto-level each3Intensityprimary/secondary vs. direct conjugatesNormalize to primary/secondary4Intensitydirect conjugates vs. direct conjugatesNormalize to brightest most specific direct conjugate
?. [Go/No Go Decision: Direct Conjugates]
Down select the D/P and concentration that yields the maximal signal to noise ratio.
?. [Go/No Go Decision: Direct Conjugates]
For the troubleshooting workflow, refer to Figure 2: Direct Conjugate Troubleshooting Workflow.
?. [Go/No Go Decision: Revalidating Direct Conjugates]
The new direct conjugate staining should have comparable localization to the old direct conjugate lot.   ABC1To Be ComparedTo Be Evaluated Normalized To2SpecificityOLD direct conjugate vs. NEW direct conjugate vs. primary/secondary antibody vs. DABAuto-level each3IntensityOLD direct conjugate vs. NEW direct conjugateNormalize to old lot
ABC1To Be ComparedTo Be Evaluated Normalized To2SpecificityOLD direct conjugate vs. NEW direct conjugate vs. primary/secondary antibody vs. DABAuto-level each3IntensityOLD direct conjugate vs. NEW direct conjugateNormalize to old lot
?. [Go/No Go Decision: Revalidating Direct Conjugates]
Maximize the signal to noise ratio.
?. [Determining Antigen Effects]
Select the treated slide with the “no dye inactivation” slide (Slide 1).  ABC1Type Of ComparisonType Of EvaluationNormalized To2Specificity“No Dye Inactivation” vs. historical images from primary down selection experiment vs. DAB imagesAuto-level each3Specificity“No Dye Inactivation” vs. dye inactivation roundAuto-level each4Difference in intensity“No Dye Inactivation” vs. dye inactivation round“No Dye Inactivation” slide
ABC1Type Of ComparisonType Of EvaluationNormalized To2Specificity“No Dye Inactivation” vs. historical images from primary down selection experiment vs. DAB imagesAuto-level each3Specificity“No Dye Inactivation” vs. dye inactivation roundAuto-level each4Difference in intensity“No Dye Inactivation” vs. dye inactivation round“No Dye Inactivation” slide
?. [Determining Antigen Effects]
No antigen effects from dye inactivation is defined as: No greater than 20% loss in stain intensity between “no dye inactivation” vs. other rounds. Stain specificity is maintained. Tissue integrity is maintained.Non specific background staining is minimal.
?. [Decoverslipping]
Take a plastic slide box and place it in secondary containment.
?. [Background Imaging]
Refer to Cell DIVE™ Imaging Manual.
?. [Determining Antigen Effects]
There should be a maximal signal to noise ratio.
?. [Primary-Secondary Staining]
Wash slides for 3x   with , using gentle agitation on shaker.
[PBS]
?. [Primary-Secondary Staining]
Wash slides for 3x  with , using gentle agitation on shaker.
[PBS]
?. [Go/No Go Decision: Direct Conjugates]
Note: If an antibody is not successful as a direct conjugate, options for including that antibody in a multiplex are as follows:Try validation with another dyeUse with secondary detection. If performing Primary/Secondary staining, an IgG block to the isotype of the primary antibodies used should be performed at a 10-fold concentration. This should occur before staining a direct conjugate raised in the same species.  For example: If isotypes of the primary antibodies were mouse IgG and Rabbit IgG, then after dye inactivating that round of staining, slides should be blocked in a cocktail rabbit and mouse serum for 30 minutes before proceeding to the next round of staining.Look for a commercial conjugateTry zenon labeling
?. [Go/No Go Decision: Primary Secondary Antibody staining ]
Note: If performing Primary/Secondary staining in the first round of staining of a multiplex, an IgG block to the isotype of the primary antibodies used, should be performed at a 10-fold concentration. For example: If isotypes of the primary antibodies were mouse IgG and Rabbit IgG, then after bleaching the first round of staining, slides should be blocked in a cocktail rabbit and mouse serum for 30 minutes before proceeding to the second round of staining.