TITLE: IHC staining
AUTHORS: Marc Bosse, Sean Bendall, Mike Angelo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is standard immunohistochemistry (IHC) procedure recommended in the Bendall and Angelo lab.</div></div>

[STEPS]
?. [Slide baking and PT module preparation ]
Bake the sections at  for  in an dry incubator    Optional :  ;     last 10 min place the slide vertical with the label side up to allow the paraffin to drip down
70 °C
Note: Some tissues or section size may need longer baking time. Recommended to bake at least 1 hour for brain tissue or TMA. This can be extended to 16 h (overnight).
?. [Slide baking and PT module preparation ]
Prepare Target retrieval solution  of target retrieval solution 10x (3-in-1) in  of Ultrapure TYPE 1 water   ABC1Total volume (mL)Volume (mL) Target retrievalVolume (mL) H2O2252.522.535054541001090
2.5 mL
22.5 mL
ABC1Total volume (mL)Volume (mL) Target retrievalVolume (mL) H2O2252.522.535054541001090
?. [Slide baking and PT module preparation ]
Put in the containers with the diluted target retrieval solution in the PT Module
?. [Slide baking and PT module preparation ]
PT Module Preheat Press RUN on digital screen and check for PREHEAT 75 on display
?. [Slide deparafination]
Linear StainerPour out reagent containers and fill with fresh reagents:  Xylene 3x, 100% Ethanol 2x, 95% Ethanol 2x, 80% Ethanol 1x, 70% Ethanol 1x, ddH2O 2x, exit stainless steel tank = ddH2O
IMPORTANT: Use fresh xylene for every deparaffination
?. [Slide deparafination]
Insert slides into slide carriersPlace the slide carrier into first xylene container
?. [Slide deparafination]
Press on Menu Check for Processing time = 30 sec, Lift bar = 976, Number of dips = 3 Set Start position corresponding to the first slide carrier position         Exemple: If the first slide carrier is at position 4, use Plus (+) or Minus (-) button to increase or decrease to get Start at: 04
?. [Slide deparafination]
Then press enter
?. [Slide deparafination]
Synchronize when the PT module temperature has reached 75˚C then Press Run on the Linear Stainer
?. [Slide deparafination]
Allow the rehydration process and wait until the slides reached the stainless steel tank and stop
?. [Slide deparafination]
Bring the stainless steel tank with the slides close to the PT module
?. [Antigen Retrieval]
Press run again and check for first  WARMUP  then  HEAT on display
?. [Antigen Retrieval]
Allow to run for 40 min at  and then cool down for approximately 50 min and reach
97 °C
65 °C
?. [Antigen Retrieval]
When the alarm sounds Stop the PT module
?. [Antigen Retrieval]
Take out the slides and let cool down at room temperature for at least
?. [Endogeneous enzyme Quenching]
Quenching Horseradish Peroxidase (HRP)
?. [Antigen Retrieval]
Prepare two Coplin jars filled with 1x PBS wash buffer
?. [Antigen Retrieval]
Open the PT Module and insert the slides in the Target retrieval solution container
?. [Endogeneous enzyme Quenching]
Make a 1:10 dilution of 30% Hydrogen peroxide H2O2  ABC1Total volumeUltrapure TYPE 1 water (mL)Hydrogen peroxide (mL)21 mL0.90.135 mL4.50.500450 mL455 mL
ABC1Total volumeUltrapure TYPE 1 water (mL)Hydrogen peroxide (mL)21 mL0.90.135 mL4.50.500450 mL455 mL
?. [Endogeneous enzyme Quenching]
Incubate for 30 min by adding  diluted H2O2 >100 µL (18 mm2) per sections  ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
?. [Endogeneous enzyme Quenching]
Prepare a Coplin jar filled with 1x PBS wash buffer
?. [Antigen Retrieval]
Transfer the slides in the first 1x PBS wash buffer and use orbital shaker set for 5 min, 70 rpm
?. [Antigen Retrieval]
Transfer the slides to the second 1x PBS wash buffer and use orbital shaker set for 5 min, 70 rpm
?. [Endogeneous enzyme Quenching]
Transfer quenched tissue/ slides to the 1x PBS wash buffer and use orbital skaker set for 5 min, 70 rpm
?. [Blocking ]
Add 100 µL of Blocking Buffer for 18 mm2  ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
?. [Pap pen]
Make sure to dry with a folded-tissue paper the slide, leaving a square of wet surface surrounding of the tissue section Note: Do not let dry the tissue section
?. [Pap pen]
Draw a square following the outside edges of the wet square with an hydrophibic ink pen (ImmEdge pen, Vector labs)
?. [Blocking ]
Place the slides in a moist chamber at  and incubate   to
0 Room temperature
?. [Endogeneous enzyme Quenching]
Proceed to step 22
?. [Endogeneous enzyme Quenching]
Quenching Alkaline Phosphatase
?. [Endogeneous enzyme Quenching]
Make a 1:100 dilution of levamisole  ABC1Total volumeImmpact diluent (mL)Levamisol (mL)21 mL0.9900.0135 mL4.9500.50
ABC1Total volumeImmpact diluent (mL)Levamisol (mL)21 mL0.9900.0135 mL4.9500.50
?. [Primary antibody]
Remove the blocking solution by tapping the slide on a side and immediately add the primary antibodyRefer to the chart for the volume of antibody to apply  ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
?. [Blocking ]
Dilute the primary antibody in antibody diluent buffer
?. [Primary antibody]
Place the moist chamber at 4˚C  , preferably in a place with low disturbance (e.g. a designated area in a cold room)
?. [Endogeneous enzyme Quenching]
Incubate for 30 min by adding  diluted Levamisole  >100 µL (18 mm2) per sections  ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)5070100150350
?. [Wash buffer]
Prepare two Coplin jars filled with 1x PBS wash buffer
?. [Wash buffer]
Transfer the slides into the first Coplin jar and use orbital skaker set for 5 min, 70 rpm
?. [Wash buffer]
Transfer the slides into the second Coplin jar and use orbital skaker set for 5 min, 70 rpm
?. [Endogeneous enzyme Quenching]
Transfer quenched tissue/ slides to the 1x PBS wash buffer and use orbital skaker set for 5 min, 70 rpm
?. [Primary antibody]
Verify stock of Antibody Diluent (3% NHS) and prepare accordingly if running low (Refer MIBI and IHC solutions protocol)
?. [Antigen Retrieval]
Verify stock of 1x PBS wash buffer and prepare accordingly if running low  AB1ReagentsQty for 1000 mL2PBS IHC Wash Buffer with Tween 20 (mL)503Bovine Albumin (BSA), heat shock treated (g)14Ultrapure (type 1) water (mL)949
AB1ReagentsQty for 1000 mL2PBS IHC Wash Buffer with Tween 20 (mL)503Bovine Albumin (BSA), heat shock treated (g)14Ultrapure (type 1) water (mL)949
?. [Endogeneous enzyme Quenching]
Prepare a Coplin jar filled with 1x PBS wash buffer
?. [Secondary antibody]
Add adequate volume of universal anti-mouse/ anti-rabbit (Vector labs, MP-7500-50)Note: 1 drop is ~40 µL  ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)50701001503503Estimated # of drops12348-9
ABCDEF1Estimated Surface area (mm)10x1015x1518x1820x2020x452Volume (µL)50701001503503Estimated # of drops12348-9
?. [Wash buffer]
?. [Seondary antibody]
?. [Seondary antibody]
Add secondary
?. [Secondary antibody]
Place sample in a humidity chamber, transfer to  , and incubate
4 Room temperature
?. [Wash buffer]
than go to step 32
?. [Chromogenic development ]
Add 1 drop of chromogenic solution (DAB) in  dilution buffer (ImmPACT DAB Peroxidase (HRP) Substrate, SK-4105, Vector lab)
1 mL
?. [Secondary antibody]
Equilibrate at   the chromogenic substrate and revealing buffer
0 Room temperature
?. [Chromogenic development ]
Take the slide from 1x PBS wash buffer
?. [Chromogenic development ]
Place the slide on a absorbant meterial (preferably white)
?. [Chromogenic development ]
At 10 s count up,  add rapidly the solution in excess over tissue section; starting with the lowest titer
?. [Chromogenic development ]
Start a count up timer and take immediatly 1mL DAB diluted solution with 1 mL pipette
?. [Chromogenic development ]
Stop at 50 s count up by tapping the side of the slide to remove DAB and putting the slide back in the wash buffer
DAB chromogenic reaction is revealed by a brown coloration in positive areas.Optimal revelation time ranges between 30s to 1 min.Rapid brown apperance (Light brown or no coloration >1 min reveals is undertitered primary antibody or negative result.Revealling time should be set constant at 40s but can be extented up to 1 min max.Optimal titer at constant development time ensure consistency when large number of slides are processed.
?. [Chromogenic development ]
OptionalChromogenic development can be performed directly under a microscope.This should be done at low magnification using a 5 x or a 10x objective. This way, it helps to cover a large field of view where positive areas can be observed. At higher magnification (e.g. 40x), it is likely that the DAB solution will contaminate the objective and cause a permanent stain if not cleaned immediately with ROR (Residual Oil Remover, EMS, 74319-35) and lens paper.This procedure can be used for single preliminary test but is not recommended for large scale.Importantly, make sure to clean the objectives and stage after use. Otherwise, DAB residues will stain permanently the surface in contact and increase deterioration of the microscope parts in the long term.
?. [Counter stain]
Prepare a 1L beaker filled with tap water in a sink
?. [Counter stain]
Set staining area by protecting counter top with an absorbant
?. [Counter stain]
Add Hematoxylin to cover all tissue sections (Hematoxylin Solution, Harris Modified, Sigma-Aldrich, HHS16-500ML)After 10s stop by dipping and rinsing in the tap water beakerReveal one slide at the time
?. [Counter stain]
Start a count up timer
?. [Counter stain]
Add 1x Scott's tap water for 10s, until blue color is revealed
?. [Counter stain]
Put in 70% ethanol
?. [Deshydration and VectaMount Permanent Mounting Medium]
Prepare the linear stainerPlace the reagents in the following order:  70% Ethanol 1x, 80% Ethanol 1x, 95% Ethanol 2x, 100% Ethanol 2x,  exit stainless steel tank = empty
?. [Deshydration and Clear Medium (toluene-acrylic based)]
Prepare the linear stainerPlace the reagents in the following order:70% Ethanol 1x, 80% Ethanol 1x, 95% Ethanol 2x, 100% Ethanol 2x,  Xylene 3x, exit stainless steel tank = empty
?. [Deshydration and Clear Medium (toluene-acrylic based)]
Let air dry for at least
?. [Deshydration and Clear Medium (toluene-acrylic based)]
Add 2 drops  of mounting media on tissue area by squeezing down the bottle gently for Cytoseal 60 or ~70 µL of ACRYTOL from a small aliquot put in a 50 mL tube
Work under a fume hood. The mounting media contains toluene as solvent.
?. [Deshydration and VectaMount Permanent Mounting Medium]
Let air dry for at least
?. [Deshydration and Clear Medium (toluene-acrylic based)]
Place a glass coverslip no. 1.5 (Thermo Scientific, Gold Seal, coverslips, #1.5, Fisher Scientific, 12-518-108A)
?. [Deshydration and VectaMount Permanent Mounting Medium]
Add 2 drops of mounting media on tissue area using a transfer pipette or a pointed cotton swab
?. [Deshydration and VectaMount Permanent Mounting Medium]
Place a glass coverslip no. 1.5 (Thermo Scientific, Gold Seal, coverslips, #1.5, Fisher Scientific, 12-518-108A)
?. [Deshydration and VectaMount Permanent Mounting Medium]
Bake at for  prior long term storage
70 °C
?. [Deshydration and Clear Medium (toluene-acrylic based)]
Let dry  under fume prior long term storage
?. [Deshydration and Clear Medium (toluene-acrylic based)]
Dip 3x the slide quickly in xylene prior mounting
?. [Deshydration and Clear Medium (toluene-acrylic based)]
Excess of mounting media can be removed by dipping back the slide in xylene and wiping excess along the slide edges