TITLE: Vezina Lab IHC Protocol
AUTHORS: Chad Vezina

[STEPS]
?. [Dewaxing, antigen retrieval and blocking.]
Heat slides to 65°C for 5 min to remove wrinkles and increase tissue adhesion.
?. [Dewaxing, antigen retrieval and blocking.]
Dewax and rehydrate slides by incubating at 25°C in Xylene (3 min, repeat twice), 100% ethanol (3 min, repeat twice), 75% ethanol (3 min, repeat twice) and 50% ethanol (3 min, repeat twice).
?. [Dewaxing, antigen retrieval and blocking.]
Prepare a 1X working solution of citrate buffer by adding 5 mL of 100X solution to 495 mL of dH20 and place into a 8”x 8”x 2” Pyrex glass dish.
?. [Dewaxing, antigen retrieval and blocking.]
Place slides in glass dish and microwave on power 50 for 20 min.  Allow slides to cool to room temperature before proceeding.
?. [Dewaxing, antigen retrieval and blocking.]
Use a kimwipe to create a dry rectangle around the tissue section.
?. [Dewaxing, antigen retrieval and blocking.]
Outline samples with hydrophobic pen, being careful to mark only the dry part of the slide.
?. [Dewaxing, antigen retrieval and blocking.]
Pipette a bead of TBSTw into the outlined region and let sit for 5 min at 25°C with no agitation so that the pap barrier can dry.
?. [Dewaxing, antigen retrieval and blocking.]
Tap off TBSTw and block for 1 hr at 25°C with gentle agitation in blocking buffer.**Pause point: samples can be stored for up to 4 hours**
?. [Dewaxing, antigen retrieval and blocking.]
Dilute primary antibody/antibodies in blocking buffer, apply to slides, and incubate overnight at 4°C with gentle agitation.
?. [Secondary antibody and coverslipping.]
Wash 5 min in TBSTw at 25°C with gentle agitation (repeat 5 times).
?. [Secondary antibody and coverslipping.]
Dilute flurophore-conjugated secondary antibody/antibodies in blocking buffer, apply to slides, and incubate for 60 min at 25°C in a light protected box.
?. [Secondary antibody and coverslipping.]
Remove secondary antibody solution and wash with TBSTw for 5 min at 25°C in light protected box. Repeat this step 7 times.
?. [Secondary antibody and coverslipping.]
Prepare DAPI working solution by combining 1.5 μl DAPI stock with 998.5 μl TBSTw, apply to slides  and incubate for 5 min at 25°C in light protected box.
?. [Secondary antibody and coverslipping.]
Wash with TBSTw for 5 min at 25°C in light protected box. Repeat this step 3 times.
?. [Secondary antibody and coverslipping.]
Add a small bead of antifade-mounting media to slide and add cover glass (Corning #2865-18). Image ASAP.**Pause point: Samples can be refrigerated (4ºC) for up to 3 days before imaging without appreciable loss of fluorescence**
?. [Solution Preparation]
Phosphate Buffered Saline (PBS, 1L of a 1X working solution)
?. [Solution Preparation]
Tris Buffered Saline-Tween-20 (TBSTw, 500 mL of a 10X stock solution)
?. [Solution Preparation]
10% (wt/vol) Roche Reagent (100 mL of a 10X stock solution)  ABC1  stock solution    final
  conc.  for 100  mL2  maleic acid    100 mM    1.2 g  3  5 M NaCl    150 mM    3 mL  4  dH2O to vol.    --    to 100 mL  5  Blocking reagent    10%    10 g  Mix maleic acid, NaCl & dH2O according to above, pH to 7.5 (note: this is a strong buffer and it is difficult to adjust pH, try using solid NaOH pellets to raise pH initially). Add blocking reagent, microwave briefly to aid solubility. Aliquot 10 mL volumes into conical tubes & store at –20°C for up to three years.
ABC1  stock solution    final
  conc.  for 100  mL2  maleic acid    100 mM    1.2 g  3  5 M NaCl    150 mM    3 mL  4  dH2O to vol.    --    to 100 mL  5  Blocking reagent    10%    10 g
?. [Solution Preparation]
Citrate Buffer (100x stock solution)1M Sodium Citrate Dihydrate (Fisher Scientific #BP327-500)Dilute with water, adjust pH to 6.0, store at 4°C for up to 6 months
?. [Solution Preparation]
Blocking Solution (100 mL)
?. [Solution Preparation]
Sudan Black B (50 mL)Make a 0.1% stock solution of sudan black by dissolving 50 μg of powder into 50 mL of 70% ethanol. Store solution at room temperature Make fresh prior to each staining procedure
?. [Solution Preparation]
Mounting Medium (10 mL)Make a n-propyl gallate 20% stock solution by combining 0.4 grams n-propyl gallate and 2 ml DMSO, prepare 100 microliter aliquots and store at -20°CMake mounting medium by combining 0.5 mL of 20X PBS solution, 9 mL glycerol, 100 microliters of 20% n-propyl gallate, and 0.5 mL H2O. Store solution at 4°C for up to 2 months.
?. [Solution Preparation]
4',6-diamidino-2-phenylindole, dilactate (DAPI) solution (10 mL of a 300 µM/ 10X stock solution).