TITLE: DAB Staining of FFPE Slides
AUTHORS: Jerelyn Nick, Franchesca Farris, Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Standard IHC staining, such as this DAB protocol, is included in the validation of new antibodies at the University of Florida Tissue Mapping Center, TMC-Florida/Zurich. This procedure includes all of the reagents necessary to perform a DAB based immunohistochemistry reaction.  DAB Substrate (3,3’-diaminobenzidine) produces a brown reaction product in the presence of peroxidase (HRP) enzyme.  </div></div>

[STEPS]
?. [Day 1 Prepare Slides for staining]
De-paraffinize and  rehydrate slides.Place slides in a xylene-compatible rack.
?. [Day 1 Prepare Slides for staining]
Immerse slide holder in first xylene station for 5 minutes.
?. [Day 1 Prepare Slides for staining]
Immerse slide holder in second xylene station for 5 minutes.
?. [Day 1 Prepare Slides for staining]
Immerse slide holder in first 100% Ethanol for 5 minutes.
?. [Day 1 Prepare Slides for staining]
Immerse slide holder in second 100% Ethanol for 5 minutes.
?. [Day 1 Prepare Slides for staining]
Transfer slide holder into  3% H2O2 in methanol for 10 minutes.
Prepare the  3% H2O2   in methanol solution fresh each day  (25ml of 30% H2O2  added to 225ml of methanol).
?. [Antigen Retrieval]
Perform heat mediated antigen retrieval using 1X citra buffer.
Alternative antigen retrieval methods can be used at this step based on individual antibody product sheet or laboratory experience/preferencve.
?. [Day 1 Prepare Slides for staining]
Immerse slide holder in 95% Ethanol for 5 minutes.
?. [Day 1 Prepare Slides for staining]
Immerse slide holder in 75% Ethanol for 5 minutes.
?. [Day 1 Prepare Slides for staining]
Immerse slide holder in 50% Ethanol for 5 minutes.
?. [Day 1 Prepare Slides for staining]
Immerse slide holder in deionized water for 5 minutes.
?. [Day 1 Prepare Slides for staining]
Immerse slide holder in deionized water for second wash for 5 minutes.
?. [Antigen Retrieval]
Remove slides one at a time from the Coplin Jar and  dip in de-ionized water to remove residual citra buffer.
?. [Blocking and Applying Primary]
Avidin blockTo make the avidin solution: combine 1mL of 1X TBST  buffer with 6 drops of Avidin from vector avidin biotin kit.
?. [Blocking and Applying Primary]
Take the slide, tap off buffer and cover section with Avidin Solution for 20 minutes.
?. [Blocking and Applying Primary]
Biotin blockTo make the biotin solution: combine 1mL of 1X TBST buffer plus 6 drops of Biotin from a vector avidin biotin kit.
?. [Blocking and Applying Primary]
Take the slide, tap off buffer and cover section with the Biotin solution for 20 minutes.
?. [Blocking and Applying Primary]
Serum blockTo make the serum block, combine  1mL of 1X TBST buffer and 20uL of Normal Serum .
The  blocking solution is prepared with from the serum from the same animal species in which the biotinylated secondary antibody is made.
?. [Blocking and Applying Primary]
Tap off biotin solution and cover sections in 1X TBST buffer .
?. [Blocking and Applying Primary]
Tap off buffer and remove the excess moisture with gauze. Cover section with serum block and incubate for 60 minutes in the hummidity chamber.
?. [Blocking and Applying Primary]
Primary antibody incubation
Prepare desired dilutions of primary antibody in Antibody Diluent (Thermo Fisher)
?. [Blocking and Applying Primary]
Remove excess serum with gauze.
?. [Day 2 Detect Antibody Staining]
Remove chamber from the refrigerator, carefully lift off and dispose of plastic bag coverslips.Wash slides with 1X TBST buffer for 5 minutes at room temperature . While washing, prepare ABC Reagent and secondary antibodies.
ABC Reagent needs to be made at least 30 minutes before it is used. To make the solution, use 20uL of reagent A and 1mL of buffer. Mix, then add 20uL of reagent B. Mix again. To make secondary solution combine 1ml of 1X TBST buffer with 15ul of serum matching species secondary is raised in.  Add 5ul of  Vector biotinylated secondary antibody.  Secondary antibody must be directed against species of origin for primary antibody.
?. [Day 2 Detect Antibody Staining]
Incubation with biotinylated secondary antibody(s).
?. [Day 2 Detect Antibody Staining]
Tip buffer of slides and remove excess moisture with gauze. Cover sections with the secondary antibody solution and incubate for 30 minutes at room temperature in a humidty chamber.
?. [Day 2 Detect Antibody Staining]
Wash slide with  1X TBST buffer for 5 minutes. Tip buffer of of slides and remove excess moisture with gauze.
?. [Day 2 Detect Antibody Staining]
Cover the sections with ABC reagent (step 8) and incubate for 30 minutes at room temperature.
?. [Day 2 Detect Antibody Staining]
Wash slides in 1X TBST buffer for 5 minutes. Slides can be held in buffer until ready for DAB staining.
?. [Day 2 Detect Antibody Staining]
DAB reactionTo make the DAB solution: add 1 drop of DAB to 1mL of Immpact Diluent.
?. [Counterstain and mount]
Counterstain.Prepare staining line in keeping with the steps below. Remove slides from water and place them into a xylene compatible slide holder.
?. [Counterstain and mount]
Dip slide holder into hematoxylin for 10 seconds.
?. [Counterstain and mount]
Rinse with running tap water by immersing slide holder into water until it is clear.
?. [Counterstain and mount]
Mount a coverslip on to the slide using a permanent mountant.
Working in the fume hood, remove one side at a time from the Xylene and mount coverslip with approximately  2 drops of Shandon Mount. Blot the edge of the coversliped slide on a  paper towel to remove excess mountant and allow to dry in the fume hood.
?. [Counterstain and mount]
Immerse slide holder into Clarifier for 20 seconds.
?. [Counterstain and mount]
Immerse slide holder into water for 1 minute.
?. [Counterstain and mount]
Immerse slide holder into Bluing Reagent for 1 minute.
?. [Counterstain and mount]
Immerse slide holder into water for 1 minute.
?. [Counterstain and mount]
Immerse slide holder into 50% Ethanol for 5 minutes.
?. [Counterstain and mount]
Immerse slide holder into 75% Ethanol for 5 minutes.
?. [Counterstain and mount]
Immerse slide holder into 95% Ethanol for 5 minutes.
?. [Counterstain and mount]
Immerse slide holder into 100% Ethanol  for 5 minutes. Transfer slide holder into a second 100% Ethanol staining station for another 5 minutes.
?. [Counterstain and mount]
Immerse slide holder into first xylene for 3 minutes. Transfer slide holder into second xylene station for another 3 minutes.
?. [Counterstain and mount]
When slides are dry, image using a brightfield microscopy sytem.  The antibody staining will appear brown and nuclei will be counterstained light blue.
?. [Antigen Retrieval]
Remove slides from slide holder and place into a plastic Coplin Jar filled with 10mM pH6.0 Na citrate buffer. Slides may be placed back to back in Coplin jar.Citrate buffer level must be over the top edge of the slides.
?. [Antigen Retrieval]
Remove the beaker and jar from the microwave and  incubate slides in the citrate buffer on the bench top for 18 more minutes. During this incubation, make 1X TBST buffer by combing 90ml de-ionized water, 10ml 10X TBS, pH 7.6, and 1ml 10% Tween-20.
?. [Blocking and Applying Primary]
Apply primary antibody solution directly over the section, cover with a plastic slip cut from a clean plastic bag surface and place in hummidity chamber.  Incubate in a 4˚C refrigerator overnight.
?. [Day 2 Detect Antibody Staining]
Wipe excess buffer from one slide.  Place the slide on to the stage of a bright field microscope, and identify an area for observing DAB development.
?. [Day 2 Detect Antibody Staining]
Cover the section on the slide with DAB solution and incubate until desired intensity develops. Once desired intensity is reached, rinse and leave in water until ready to counterstain.
Collect DAB waste for hazardous chemical disposal.
?. [Antigen Retrieval]
Place slides in a humidity chamber and immediately cover secttions with 1X TBST buffer.  Equillibrate for 5 minutes
?. [Antigen Retrieval]
Place the Coplin Jar in a 400ml beaker filled with water leaving a centimeter of space between water level and cap bottom edge. Leave the cap loose, but held in place so that steam will not remove it.
?. [Antigen Retrieval]
Place beaker / Coplin jar  assembly into microwave oven and heat for 7 minutes at 50% power.