Calcium channel IF

[hobbes]

Hi,
I have problems with my IF on frozen rat brain sections for L-type voltage-gated calcium channel. The Ab is rabbit, polyclonal from Sigma
I use the following protocol
2x5min PBS
antigen retrieval (heating the slides in 0.2% citrate buffer pH=6.0 for 10 min at 800W)-I could not get the staining if I don't do this, even though these are frozen sections
2x5min PBS
1%BSA+0.3% Triton (I also tried 5%serum+0.3% Triton) 1h@RT
rabbit Lc-type (Cav1.2) Ab (1:500 in 1%BSA) overnight@4C
4x5min PBS
donkey anti-rabbit-Alexa 488 1:200 1h@RT
4x5min PBS
1%BSA+0.3% Triton (I also tried 5%serum+0.3% Triton) 1h@RT
mouse anti-GFAP in 1%BSA (1:200) overnight@4C -this works, the problem is only with L-type Ab
donkey anti-mouse Alexa 555 (1:200) 1h@RT
4x5min PBS
DAPI 20min
4x5min PBS
mount
the problem is that I get the nuclear staining with L-type Ab, which is most probably totally wrong.
Should I omit Triton, since it is a membrane-bound epitope? But I have read a lot of articles where they use Triton for staining the calcium channel..so Im confused
Please help
Thx

[richard03]

Triton X-100 is used to permeabilizing cell membranes therefore enhance antibody penetration and improve staining. It dose not dissolve or destroy membrane.

I use Triton X-100 for membrane protein staining routinely and have not noticed any significant issues.

The nuclear staining pattern you observed may be due to autofluorescence or due to using antigen retrieval steps.

[athurart09]

I use Triton X-100 for membrane protein staining routinely and have not noticed any significant issues.

me too