What’s all the Fos about? (staining protocol)

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. Click on images to biggify.

Fos 1:250 primary antibody dilution, without amplification:

Here you can clearly see some Fos+ cells, especially in the dentate gyrus, where signal is always greatest. But there aren’t many cells there, or in other regions. We don’t expect that many cells, because of sparse coding in the hippocampus, but still…

Fos 1:250 primary antibody dilution, with amplification:

Now we’re talking! Fos everywhere!

Fos 1:5000 primary antibody dilution, with amplification:

Basically the same as 1:250 except possibly less background staining, better signal to noise, and save $$$.

c-Fos Fluorescent Immunohistochemistry Protocol

Day 1:
1) 3 x 5 min wash in PBS
2) 30 min wash 10% PBS-Triton X (TX) with 3% horse serum
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:
1) 3 x 5 min wash in PBS-TX
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)
3) 3 x 5 min wash in PBS
4) 30 min Blocking Solution at 5%
a) To make blocking solution:
– 40 mL PBS
– 0.2g blocking powder (Perkin Elmer, FP1020)
– Warm to dissolve then cool to room temp before use
5) 1:100 Streptavidin Horseradish Peroxidase (Perkin Elmer, NEL750) in Blocking Solution for 1 hour
6) 3 x 5 min wash in PBS
7) 30 min 1:2000 NHS-Rhodamine (Fisher, PI-46406), in PBS w/ 1:20,000 H2O2
8) 2 x 5 min wash in PBS
9) 1 x 5 min incubation 1:1000 DAPI in PBS
10) 4 x 5 min wash in PBS

Staining Recommendation: Incubate any additional primary antibodies separately from amplification Day 2 after adding the Rhodamine (step 7) and completing 3 subsequent PBS washes.

Acknowledgements: Amplification protocol was devised by PhD student Shaina Cahill and undergrad student Angela Martinovic. Dilution fidgeting performed by MSc student Alyssa Ash. Wisdom, guidance and praise contributed by Jason Snyder.

This protocol is also published on Figshare, an open access medium to rapidly share scientific information:

Martinovic A, Ash A, Cahill SP, Snyder JS (2017) c-fos immunohistochemistry protocol. Figshare: https://doi.org/10.6084/m9.figshare.5229898.v1