Re: [Histonet] Re Protocol for DAPI staining on paraffin sections

[Hobbs, Carl via Histonet]

Dear Alida,
That is a good point.
I probably have only done HOECHST on Pwax sections after they have been 
heat-Antigen retrieved ( HIER)
However, H ( or DAPI) works on fixed cell monolayers: they don't get HIER 
treatment)
Nor do frozen sections ( well, some do) and the HOECHST/DAPI works fine on both
However, perhaps you can just take a Pwax section to water and apply HOECHST 
for 10 mins, wash off, mount in anti fade aq. mountant and have a look down a 
Fluorescence scope?
If you can wait, I will check this next Monday ( put H on a non HIER'd Pwax 
section  and get back to you by Monday later)

Unless, whoever that is doing the fluorescence DAPI/HOECHST nuclear staining is 
actually doing it on Pwax sections that have been HIER-ed?

Best wishes

Carl



Carl Hobbs FIBMS
Histology and Imaging Manager
Wolfson CARD
Guys Campus, London Bridge 
Kings College London
London
SE1 1UL
 


020 7848 6810
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[Histonet] Re Protocol for DAPI staining on paraffin sections

[Hobbs, Carl via Histonet]

Use DAPI/HOECHST at the same concentration you use for cells/FS
I use Hoechst H33258 Sigma 10mg/ml soln
I dilute by adding 0.5 microL to 100 microL buffer then add 1/100 to Alexa 
secondaries ( final 1/20K diln factor)
Some incubate in HOECHST or DAPI after secondary ab incubation, for IF
Sure, if only "staining" nuclei, incubate in 1/20K Hoechst in same buffer
This will vary for each lab
Or, buy mounting medium that contains DAPI

Good luck
Carl

Carl Hobbs FIBMS
Histology and Imaging Manager
Wolfson CARD
Guys Campus, London Bridge
Kings College London
London
SE1 1UL

020 7848 6813
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