c-fos Immunohistochemistry Protocol

<b>Introduction</b>Many studies on memory and learning utilize the immediate early gene c-Fos as an indicator of recent synaptic activity. By quantifying c-Fos positive neurons, we can compare activity across different populations in the brain, and analyze co-labelled neurons with other useful cell markers such as BrdU and Doublecortin.Our lab recently devised an amplification protocol for c-Fos stains that specifically increases the intensity of the signal via a Biotin-Streptavidin-Rhodamine complex (see below). We have demonstrated this amplification protocol is effective over a wide range of primary antibody dilutions, with strong signaling from 1:250 to 1:5000 dilution. Basically, our amplification protocol is a fluorescent dream come true. But don’t just take our word for it, check it out for yourself. Dilutions and amplification conditions are stated in the image filenames.<b>Protocol</b>Day 1:<br>1) 3 x 5 min wash in PBS<br>2) 30 min wash 10% PBS-Triton X (TX) with 3% horse serum<br>3) Add primary antibody for 3-day incubation period (tested range of 1:250 to 1:5000 dilution of c-Fos antibody (Santa Cruz, sc-52-G) in 10% PBS-TX w/ 3% horse serum)Day 2:<br>1) 3 x 5 min wash in PBS-TX<br>2) 1 hour at room temp Secondary Antibody (biotinylated anti-goat 1:250, Jackson Immuno #705-065-147, in 10% PBS-TX w/ 3% horse serum)<br>3) 3 x 5 min wash in PBS<br>4) 30 min Blocking Solution at 5%<br>a) To make blocking solution:<br>- 40 mL PBS<br>- 0.2g blocking powder (Perkin Elmer, FP1020)<br>- Warm to dissolve then cool to room temp before use<br>5) 1:100 Streptavidin Horseradish Peroxidase (Perkin Elmer, NEL750) in Blocking Solution for 1 hour<br>6) 3 x 5 min wash in PBS<br>7) 30 min 1:2000 NHS-Rhodamine (Fisher, PI-46406), in PBS w/ 1:20,000 H2O2<br>8) 2 x 5 min wash in PBS<br>9) 1 x 5 min incubation 1:1000 DAPI in PBS<br>10) 4 x 5 min wash in PBSStaining Recommendation: Incubate any additional primary antibodies separately from amplification Day 2 after adding the Rhodamine (step 7) and completing 3 subsequent PBS washes.