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Author Topic: C fos staining troubleshooting  (Read 6336 times)

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Offline SLee

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C fos staining troubleshooting
« on: September 09, 2011, 01:32:22 PM »
Hello,

I am staining mouse brain sections for c-fos using a colorimetric (ABC/DAB) protocol.  The sections are mounted 10micron frozen fixed sections. I've been having trouble optimizing my staining protocol and have been getting very low signal of c-fos. My protocol is as follows:


Thaw out slides at Room temp for 15 min. Dry out slides in 37 degree incubator for 15
4% PFA 5 min
PBS 3x2min
.3% H202 in MeOH 20 min
PBS 3x5min
Glycine 10min
PBS 3x5min
SDS 10 min
1:100 c-fos primary Ab (Santa Cruz) Overnight at 4 degrees (about 16 hours)

Next day:
PBS 3x5 min
1:500 biotinylated secondary Ab (Vector labs) 2 hours at Room temp
PBS 3x5 min
Vector ABC kit solution 1 hr.
PBS 3x~20 Sec
DAB ~2 min

At first, I got no signal, so I thought maybe the c-fos was getting degraded when I dried my slides in the 37 degree incubator. When I took that step out, I did notice some better signal, but it is barely detectable (nowhere good enough for quantification). I was wondering if anyone had any suggestions to improve the signal for c-fos. Thanks!

C fos staining troubleshooting
« on: September 09, 2011, 01:32:22 PM »