Poor IHC results...need help

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vilsy
vilsy's picture
Poor IHC results...need help

Hi,
I am trying to look into some GABAergic interneurons in the thalamus. I fix the animal with 4% PFA overnight and then block the tissue in OCT medium and section on a freezin microtome at 40 microns.
While my staining for NeuN has been giving me beautiful results, I cannot say the same about GAB65/67 and Parvalbumin staining. GAD staining is very very weak while parvalbumin does not seem to work. I was discussing this with a coulleague and he suggested me to use glutaraldehyde instead of paraformaldehyde but could not give any reason for it.
I did my research on the internet and since both GAD and Parvalbumin are not membrane associated, I do not understand what can change? Any idea guys on what I can do to improve the results of my stainings.
 
Cheers,
Vilay

Arvind Singh Pundir
Arvind Singh Pundir's picture
Hi Vilsy

Hi Vilsy
nice to have you back after a gap, regarding your IHC can you mention the dilution of your primary Ab and your breif protocol i have been doing IHC for parvalbumin for the past 4 years on human Auditory cortex and its working very fine , i am using the SWANT Ab which i seem are pioneers in calcium binding proteins, for me even 1:10000 dilution of parvalbumin works great , changing the fixative i seem is not the issue as PFA should work well so , it will be helpful if you mention your brief protocol .........

Fraser Moss
Fraser Moss's picture
Can you give a little more

Can you give a little more detail about your fixing protocol please Vilsy?
Are you just plopping the brain into PFA after you remove it, or are you rapidly perfusing the animal and then removing and fixing the tissue?  If you don't get the animal perfused quick enough, the quality of the tissue can be really poor for staining.  NeuN lights up so much that it can still look apparently ok in bad sections until you put it side by side with more specifically localized labels.

Also the quality of the paraformadehyde can make a difference.  Try and use EM grade PFA and even get the 4x (16%) pre-dissolved stuff which you can dilute from sealed aliquots fresh for each use.
Beware using glutaraldehyde as there can be issues with autofluorecence.

Guest (not verified)
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Hi,
From my experience about 40%  of  the commercial antibodies do not work like they state in the product information. 
1)       Select an AB that a friend, colleague or paper has used successfully in the same fixation protocol that you are using.
2)       Some polyclonal antibodies have been raised against; non fixed or glutaraldehyde or paraformaldehyde fixed antigens. The antigen is altered by the fixation, and if the antibodies have been raised against the glut or para fixed antigen you should use the same fix to get th best results.  Each of the fixatatives alters the antigen in specific ways.
  

Arvind Singh Pundir
Arvind Singh Pundir's picture
to what extent does different

to what extent does different fixatives alter the IHC results differently, i mean that if a tissue is fixed in PFA and the other in Glutraldehyde so does the protocol differ if we are using the same conditions of Ab and dilutions etc. for the same tissue in both cases except the antigen retrieval ......

Guest (not verified)
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Hi Pundir,

Hi Pundir,
The antigen antibody have to be matched first after that there are just the normal IHC proceedures. 
Some antibodies will not work at all if fixed or fixed with the wrong fixative.  (You can also try ethanol and acetone fixation).
All the proceedures are the same.  You have to do a dilution curve for primary and secondary antibody. You have to adjust the incubation time for section thickness.  You need to block appropriately.
If all else fails try antigen retreaval.  I believe it is a last resort. 1) it can make the tissue look terrible. 2) it can give false positives that are very hard to control for.  (if the antigens are smashed up, epitopes that look like the origonal antigen could be produced, giving you false positives).  You need to run positive and negative brain tissue to be sure this is not happening. In the past I have rejected papers that I was reviewing because the appropriate controls were not incuded.
OCT

NextAdvance
NextAdvance's picture
Another fixative you can try,

Another fixative you can try, which we commonly use in my lab if we stain PFA-fixed tissue and it doesn't work, is 3% TCA.  Just make sure you don't fix tissue in TCA for more than a few hours (3, max).
I agree with OCT that it could very well just be the antibody.  Has the antibody been sucessfully used for IHC in the literature, or do you know anyone that has sucessfully used it?
Also, I avoid fixing with Methanol or Acetone when I'm doing microtomy, as the tissue generally needs a stronger fixative in order to hold up well.
Good luck!
-Carlton