[Histonet] PFA Fixed tissue not sticking to slides?

[Michelle Chang via Histonet]

Good charged slides or subbed slides will work.

In regards to the fixation, I personally find the morphology with 4% PFA O/N 
followed by 30% sucrose better than doing them simultaneously.

Michelle
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[Histonet] FLO.30640:

[J B via Histonet]

CAP says that I need to have a procedure for Flow cytometry interpretation.


I have asked our reference laboratory for the procedure & they will not
help. Does anyone have a flow cytometry interpretation procedure that they
can share?

Sincerely,

JB
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Re: [Histonet] PFA Fixed tissue not sticking to slides?

[Hobbs, Carl via Histonet]

Hi Jenny.
Can you use Pwax embedding as an alternative?
H will always be better on Pwax sections. 
IHC will also be in many instances.
What abs are you probing with?
Single/double-labelling?



Yesyou are fixing for too long ( for frozen sectioningfor Pwax you can 
fix your specimens for a week and still not suffer IHC-problems: after HIER 
they will or will never work, depending upon ab)
PFA( Formalin) fixed sections are well-known for not being as adherent as 
unfixed frozen material.

Why not freeze unfixed?
No sucrose required ( all that does is allow for slow freezing without, in most 
cases, formation of ice-crystals: good snap freezing does not need sucrose 
pretreatment)

If you are after good morphology then Pwax is the way to go, imho.
If you have to cut PFA-fixed then frozen tissues, after mounting on slides 
stick them in a 50C oven for 2hrs.
Use Superfrost Plus slides.
Then use or, store at 4C for a week or freeze.
For the latter 2, ALWAYS take out of fridge/freezer and immediately place under 
a fan/operating fume hood.
Do not allow condensation to form.
Orplace them immediately into that same 50C oven for an hour.
Do not do this to unfixed frozen sections!

Sure, check out EC's excellent advice ( as always)  re slides.

Interestedly,
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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