your first question is not clear.

For your second question: It is recommended to use serum of species where secondary antibody is generated. Otherwise, FBS is universal.

Dear Dr Naseer Ahmed

My feedback to your questions is as follows:

1.    It looks like you are trying to compare the expression level of a receptor in horizontal and vertical sections from two different organs (e.g. intestine vs stomach). I do not see any reason why that is not possible? It is the sub-cellular localization of the receptor which matters the most; the orientation of the section would be a secondary criteria. However, if you are really concerned about it, it would not hurt to run pilot testing on horizontal/vertical sections from both tissues to see which orientation gives you the best outcome/results.  

2.    About blocking step, it is advisable to use a blocking buffer from a serum of the animal which is the host of the secondary antibody. For example, if you are using a Goat-anti Mouse secondary antibody (for Mouse anti-Rat primary), 5% Goat serum in PBST (PBS + 0.4% Triton X 100) is what I would suggest for use as a blocking buffer. 30-60 minutes of blocking is pretty standard for IHC/IFF analysis of most of the protein targets.

I hope this is helpful but please feel free to share if there are any counter questions or if you need any other feedback.

Best Wishes

Subhash

Dear Dr Subhash,

Thank you for your kind response.

Its really useful and helpful for me.

About sections, as i am not very expert in it. So, actually i am checking different chambers of heart. For some i have horizontal sections and for some specimens i have vertical sections. But my purpose is just to see receptor expression.

As you said its subcellular level, so will it work?

Thanks alot.

Dear Dr Subrata,

Thanks for your kind reply. How much % of FBS or antibody specific serum should ideally use?

If someone use 10% and someone 1%, how much can be good or bad?

Thanks

Dear Dr. Mohammed,

Thanks. I try to use it.

Thanks.

Immunofluorescence is not the ideal method to evaluate protein expression. It would be best if you could do RT-PCR (to evaluate mRNA levels) or immunoblot analysis of your samples (using a protein such as actin or tubular as a "control" for protein loading). However, if your sample is heterogeneous, then immunofluorescence may be better suited to examine specific cell types.

The orientation of the section does not matter. What matters however is to have a reference standard (internal control) that you can use to ratio your signal. This is because there can be individual differences in the staining intensity between sections, that can depend on thickness, handling, etc...so if you can, take a protein that you know should NOT change between your different specimens, and even more useful, if it is localised in a similar cellular substructure as your receptor Then optimize antibody dilution for both antigens, to get the highest possible dilution of antibody that gives a good signal. This is because if you want to use IF as a semi-quantitative assay, your signal must be linear with concentration, so you cannot saturate the specimen with antibody (you would get strong signal even if you had half the amount of antigen). Then do a double IF, and evaluate pixel intensity in the 2 channels, the one of your receptor and the one of your control protein, in the same or neighbouring areas (using a program such as ImageJ. Then express your data as ratios of signals (receptor/control). Do it for many regions, and plot averages with SD.

Dear Dr. Sandra,

Very useful informations.

Yes, I am doing  RT-PCR too.

But actually I want to know are these receptors express in VSMC or endothelial cells. I used CD31, but it is not specific :( . I do not know which marker should I use?

Thanks again for all great help and information.

Regards,

Naseer

Dear Prof. Sandra,

Kindly, can you suggest some endothelial marker more specific?

Thank you very much.

Best regards,

Naseer

For endothelial cells you can use anti-PECAM antibody. Or VE-cadherin.For smooth muscle cells you can use the smooth muscle actin isoform. Otherwise look up articles by Elisabetta Dejana's group for endothelial markers.

Dear Prof. Sandra,

Thanks.

Best regards,

Naseer