Active Caspase-3 Immunofluorescence 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)
- AlexaFluor™ 594 goat anti-rabbit IgG (Molecular Probes, #A-11037, Eugene, OR)
- AlexaFluor™ 488 isolectin IB4 (Molecular Probes, #I-21411, Eugene, OR)
- AlexaFluor™ 488 glial fibrillary acidic protein (GFAP, Molecular Probes, #A-21294, Eugene, OR)
- Dark humidity box
- Prolong® Antifade Kit (Molecular Probes, #P-7481, Eugene, OR)
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 distilled water for 5 min.
- 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.
- Wash the slides in PBS 3X2 min. Prepare 10% goat serum by diluting normal goat serum into 1% BSA in Auto buffer. The sections are incubated for 30 min. This step appears to reduce background fluorescence.
- Prepare the secondary antibody by diluting Molecular Probes AlexaFluor™ 594 goat anti-rabbit 1:500, into 25% normal goat serum prepared with 1% BSA/Auto buffer. The slides are incubated for 30 min in secondary antibody then are washed 3X2 min in PBS.
- In preparation for “counterstaining” with IB4/DAPI or GFAP/DAPI the sections are incubated in PBS with an additional “pinch” of Ca2+, Mg2+, and Mn2+ and 0.1% Triton X-100 for 15 minutes. The slides were rinsed through a quick change of PBS then were incubated in either 1:200 IB4/1:10,000 DAPI, or 1:200 GFAP/1:10,000 DAPI for 30 min. The slides were then washed 3X2 min in PBS. This step detects microglia and astrocytes, respectively, while labeling nuclei blue.
- Prepare the Prolong® antifade mounting media according to directions. Allow the slides to dry approximately 3/4 to completion. Coverslip with Prolong®, taking care to remove any bubbles formed beneath the coverslips.
Although the slides can be viewed immediately, the Prolong® datasheet says that antifade protection is best after the mounting medium has been allowed to dry overnight.
The active caspase 3 appears red, microglia or astrocytes green, depending on the section being observed, and nuclei are blue.