IHC of paraffin sections

To deparaffinize place slides in:

To re-hydrate place slides in:

Antigen retrieval steps: To remove methyl-bonds between proteins that were initiated during formaldehyde fixation, in order to open up epitopes.

Boil slides for 30 minutes in unmasking solution (purchased from Vector Laboratories, probably either a citrate buffer or TRIS-HCl) (1:100 or 15 mls into 1.5 L) via microwave (make sure special care is taken so the unmasking solution stays above the slide level as some of the solution will boil off). Let cool to room temperature by placing beaker in ice. NEVER LET SLIDES DRY OFF AFTER THIS STEP
1x PBS - 5 minutes

Block all endogenous peroxidases in the tissue by placing slides in:

Block non-specific binding of antibody

Place slides in humidity chamber. (A humidity chamber = any enclosed box with wet paper towels, for example, a plastic box 4 cut-off pipets, which are used to raise the slides off of the floor of the chamber. Both below and on top of the pipets includes a layer of paper towels. Water is poured into the chamber so some small puddles form on top of the paper towels.)
Blocking step - use an ImmunoEdge Pen and draw a box around the tissues of each of the slides being stained (the smaller the box, the better). Add around 100 µL of 2.5% sheep serum in PBS to each slide. The box drawn by the pen will keep the liquid from dispersing across the entire slide. Incubate at room temperature in the humidity chamber for 1 hour. Alternatively, place ~100 µl of diluted (2 drops (90 µl) into 2.5 ml PBS or TBS) M.O.M. Mouse Ig Blocking Reagent (Vector laboratories) on the slide and incubate 1 hr at Room temp.

Note: Vector Labs recommends using the exact timing indicated below for optimal staining and avoidance of background signal.

Wash slides 2x in PBS for 2 min.
Place ~100 µl (enough to cover tissue) of M.O.M diluent (600 µl stock into 7.5 ml PBS or TBS) on each tissue section, incubate 5 min at room temp.

Primary Antibody Staining

Tip off excess M.O.M. diluent. Dilute primary Ab in M.O.M. diluent. (For NCL-Ki67, dilute 1:200). Place 100 ��l of 1�� Ab solution on tissue sections and incubate overnight at 4°C in humidity chamber (Note: this step is variable and can be adjusted for better staining depending upon the primary antibody being used). While Ab is incubating, prepare the VectaStain Elite ABC reagent, so it can stand for 30 min before using.
- SB11 antibody from R&D Systems (Catalog # MAB2798) dilute to 2.5 µg/ml Ki67 antibody from NCL dilute 1:200
Wash 2x in PBS for 2 min.

Secondary Antibody staining

Place 100 µl of diluted M.O.M Biotinylated anti-mouse IgG (10µl stock into 2.5 ml of MOM diluent) onto tissue sections and incubate 10 minutes RT.
Wash 2x in PBS for 2 min.

HRP avidin-biotin addition

Place 100 µl VectaStain Elite ABC (avidin/biotin-HRP complex) reagent and incubate for 5 min. (2 drops of reagent A to 2.5 mls of PBS, mix, then add 2 drops of Reagent B and mix, let stand 30 min before using).
Wash 2x in PBS for 5 min.

Apply Diaminobenzidine (DAB) substrate to visualize antibody staining

Prepare fresh substrate (DAB from Vector Laboratories) by adding 2 drops of Buffer Stock Solution to 5 ml distilled H2O, mix well. Add 4 drops of DAB Stock Solution and mix well. Add 2 drops of H2O2 solution and mix well.
Place 100 µl on tissue sections and incubate for 2 to 10 minutes.
Wash in H2O 5 min.

Stain with Hematoxylin (following Harris procedure)

70% Ethanol - 5 minutes
Harris’ Hematoxylin acidified (filter before use) - 2 minutes
Water (rinse under tap about one minute or until the blue stain no longer runs of the slides)
Acid Alcohol (1% HCl in 70% ethanol = 1 ml conc HCL (12 M) in 100 ml 70% EtOH) - 5 seconds
Water - 10 second dip
Ammonia Water (1% ammonium hydroxide in water (1 g ammonium hydroxide in 100 ml H2O) - 10 seconds
Water (rinse under tap 1 minute).

Dehydrate tissue sections