calcium channel

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tigrich
tigrich's picture
calcium channel

Hi,
is anyone using antibodies for voltage gated calcium channels? I am doing IHC on paraffin-embedded rat optic nerve and have trouble for antigen retriveal. I have used cooking in 0.2% citrate buffer in microwave so far.
any other suggestions? Have you used trypsin?because it was not good for me

scolix
scolix's picture
I have protocol for antigen

I have protocol for antigen retrieval if u have cryosections. Anyway if u need it, I could send it to you.

Fraser Moss
Fraser Moss's picture
Here's a protocol i used to

Here's a protocol i used to stain VDCC subunits in paraffin embedded brain sections

Tissue processing and staining

Slides were brought to water by serial immersions for 2 min in Xylene (×2), 100% industrial methylated spirits (IMS)(×2), 90% IMS, 70% IMS and then rinsed in running tap water and finally left to stand in distilled water. The limits of the tissue on the slide were marked with a Pap Pen (Daido Sangyo Co Ltd., Japan). To lower the threshold of antigen detection, tissue slices were processed according to the microwave antigen retrieval process (McKee et al., 1993; Shi et al., 1991). This removes cross-linking between antigens and fixative reagents. Sections were immersed in 0.01M citrate buffer pH6 (HDS supplies, Aylesbuy, UK) and heated at 100°C for 5min in a molecular pathology microwave (BioRad). Sections were left to stand for 5min, boiled for a further 5min at 100°C and left to stand for 2min. Endogenous peroxidase activity was blocked by incubation with 3% H2O2 (Sigma) in water for 20min. Slides were then washed in running tap water for 5min then placed in distilled water for 5 min and finally washed for a further 5min in running distilled water. Non-specific binding of Abs was blocked by incubation with 5% milk powder, diluted in PBS and 0.1% Triton X-100 for 30min. Sections were incubated overnight with primary antibody diluted with 3% goat serum and 0.1% Triton X-100 at 4°C in a moist chamber. Slides were then washed 3 × 5min in PBS and the secondary biotinylated rabbit link antibody applied (1:20 dilution, Biogenex, Wokingham, UK) and incubated for 20min at room temperature. Slides were then washed 3 × 5min in PBS and incubated for 20min with Horse Radish Peroxidase (HRP) - Strepavidin-conjugated antibody (Biogenex, prediluted (1/20)). Slides were then washed 3 × 5min in PBS. 3,3',5,5' Diaminobenzidine Tetrahydrochloride (DAB) solution was made up according to Vector kit SK-4100, and incubated with sections for 2-10 min. The reaction was stopped by plunging slides into distilled water. Slides were dehydrated by immersing for 2min each in a series of 70% IMS, 90% IMS, 2 × 100 % IMS and 2 × Xylene. Slides were mounted in Xylene and cover-slips attached using an automated cover-slipping machine.

tigrich
tigrich's picture
thanx for the protocol

thanx for the protocol
Have you used antibodies from Alomone? How did you use the control antigen for preadsorbtion of the antibody?adding antigen to antibody solution in ratio 1:4 and incubating for 1h/RT and then centrifuge and use the supernatant-that is what I was told by techical support-but I dont understand it-dont the antigen-antibody complexes get to the pellet? What's the use then of the supernatant

Fraser Moss
Fraser Moss's picture
The controls I always run are

The controls I always run are:

1. Sections in which PBS was substituted for the primary antibody were run in every experiment.

2. Sections were incubated with primary Ab that had been preabsorbed with 100μM of specific synthesis peptide overnight at 4°C prior to the experiment.

My former lab always tried to avoid the Alamone VDCC Ab's but they may have made better ones since I worked on those channels (It's been 4 years)! We always ended up generating our own Ab's to get the best results.

Fraser Moss
Fraser Moss's picture
If the alamone Ab's dont work

If the alamone Ab's dont work out - check out the ones listed here

http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Cell_Signaling/Product_Highlights/Ion_Channel_Solutions.html?sa_campaign=email/promo/cid5130/2006-06-21/ion_channels

Just double check Sigma are not reselling Alamone's reagents.