[Histonet] Lipofuscin and counterstaining DAb IHC

[Tony Henwood]

Csaba,

I can't answer the fluorescent question but for light microscopic IHC I would stain for the NeuN using peroxidase-DAB followed by a PAS for lipofuscin followed by Mayer's or Harris's haematoxylin.

Result is Brown, pink, blue

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of szigcs <@t> bio.u-szeged.hu
Sent: Thursday, 2 June 2011 4:44 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Lipofuscin and counterstaining DAb IHC

Dear Histonetters!

I have human brain samples (age 70, HUntington disease) and my goal is to quantitate changes in the number of neurons/glia cells with nuclei staining.
I use NeuN IHC and Höechst. My first question is:
  - Throughout the whole sample there are a lot of lipofuscin signal fading the NeuN signal, we are not able to make any microphtograph for cell counting.
I tried Sudan black staining and Sigma autofluorescent reagent with the same result. Could you advise some tips and tricks to avoid this?

An alternative solution could be DAB IHC for NeuN and  counterstaining with nucleus staining.
Second question is:
- what type of counterstain do you recommend to be able to identify the IHC and normal nucleus staining in the same section?

Thank you in advance, best regards
Csaba





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