Millipore Technical Publications
Protocol: IHC Paraffin, Enzymatic Detection | ||
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Active Caspase-3 Immunohistochemistry Using Paraffin Sections
Reagents/Supplies:
- Xylene
- 95% EtOH
- 0.0lM Citrate Buffer, pH 6.0
- PBS, pH 7.4
- Normal goat serum
- “Auto Buffer” (0.01M Tris, pH 7.4, 1 mM CaCl2, 1 mM MgCl2, 0.1% Triton X-100)
- 1% BSA in “Auto Buffer”
- Decloaking Chamber (pressure cooker used for antigen retrieval) (Biocare Medical, LLC, Walnut Creek, CA)
- PAP™ Pen (Kiyota International, Elk Grove, Illinois)
- Rabbit Isotype Control (Zymed Laboratories, San Francisco, California)
- Active Caspase-3 antibody (rabbit) (Chemicon Int., #AB3623, Temecula, CA)
- Vectastain® Elite Kit (Rabbit IgG) (Vector Laboratories, Burlingame, California)
- Dark humidity box
Procedure:
All procedures are performed at room temperature in a light protected humidity box unless otherwise noted.
- Deparaffinize the slides by immersing in Xylene for 2 X 5 min, with occasional agitation. Rehydrate the slides by passing them through a graded series of ethanols in the following order for 5 min each: 95, 70, 50, & 25%. Wash the slides 2 X 5 min in PBS.
Note: at no time during the immunohistochemical procedure should the sections be allowed to dry out, as a high background may result. - Place the slides in a plastic slide rack in a plastic Coplin jar and cover the slides with 0.01 M Citrate Buffer. Place the slides in the decloaking chamber and set for three minutes, taking care to turn past 20 minutes and back to three. Once antigen retrieval is complete (light will switch from heat to warm) take the slides out of the decloaker and allow them to cool for 20 min at room temperature. Wash the slides with cool tap water for 5 min then transfer to PBS.
- Remove each slide, one at a time, label and circle the sections with a PAP™ pen, to minimize the amount of reagents needed. Immediately after circling the sections place the slides in the humidity box and cover the sections with PBS.
- Prepare 10% normal goat serum by diluting normal goat serum into 1% BSA in Auto buffer. Incubate the sections in 10% goat serum for 30 min.
- Make a 1:100 dilution of Chemicon polyclonal anti-Active Caspase-3, diluting into 1% BSA in Auto buffer. Gently tap the normal goat serum from each section and replace it with primary antibody. Apply 2 drops of Zymed rabbit isotype control to sections that were designated negative control. Allow the slides to incubate overnight at 4°C, then wash 3X2 min in PBS.
- Make the secondary antibody dilution by combining 3 drops normal serum into 10 mls of PBS and adding 1 drop of biotinylated anti-rabbit IgG from Vectastain Elite Kit. Apply 50-100 µl of secondary antibody dilution to each slide and allow them to incubate for 30 minutes.
- At this point prepare the Avidin-Biotin Complex so that it can sit out for at least 30 minutes prior to use. To make ABC combine 5.0 mls dH2O, 2 drops of Vectastain reagent A and 2 drops Vectastain reagent B then mix well. Remember the ABC must sit 30 minutes before using. Wash the slides in PBS 3X2 minutes.
- Incubate the sections with ABC reagent for 30 minutes. Wash with PBS 3X2 min.
- Make the Peroxidase Substrate using the Vector kit. First, add two drops of Reagent 1 (Vector Peroxidase DAB Substrate Kit) to 5 ml of distilled water. Mix well. Then add 4 drops Reagent 2 and mix well. Finally, add 2 drops of Reagent 3 and mix the solution well. Protect from light.
- Place 50-100 µl of DAB peroxidase substrate on each tissue section and allow it to incubate for 4-10 minutes (staining progress can be monitored under the inverted scope), then wash the slides with distilled water 3X2 min.
- Counterstain the slides by applying 1-2 drops of hematoxylin QS to each section. Allow the hematoxylin to incubate 1 min then wash in distilled water. (Alternatively, routine Cresyl Echt Violet stain for Nissl substance makes a nice counterstain for DAB).
- Allow the slides to dry. Apply GVA mount to the slides place coverslips on the slides. Take care to remove any bubbles formed beneath the coverslip.