[Histonet] frozen sections - lung

[Melissa Mazan]

Hi all,
I am writing to request help with frozen sections.  We do most of our 
work in FFPE tissues and are just starting on frozens.  I'm using a 
protocol posted, I believe, by Gayle Callis a while ago.  Briefly, we 
anesthetize the mouse, free the trachea of surrounding tissues, place a 
20 g catheter through the trachea, then open the chest and free the lung 
tissue, and transect the abdominal aorta.  We perfuse through the left 
side of the heart first with around 10 mls of PBS, then with 10 mls of 
4% paraformaldehyde.  This usually turns the lungs white.  Then, we fill 
the lungs with one vital capacity of OCT, clamp the trachea, and 
carefully dissect the lungs free - then either snap freeze the whole 
lung or lung lobes in OCT in a tissue tek cassette, and store at -80. 
I then make slides - about 10 microns thick - on star plus slides  -  
and let air dry at least one hour, or overnight, then post fix them 
either in acetone at -20 C for 10 minutes, or in paraformaldehyde for 10 
minutes.
The tissues look very nice on H and E - good alveolar and airway 
structure, quite nice quality cells and scaffolding.  However, my 
immunostaining is NOT coming out as well as it usually does on paraffin 
sections.  Do you make modifications to your protocols for paraffin? 
(usually permeabilize with either TBST or PBS-Tx, block for one hour, 
primary diluted in same buffer for either 1 hour at RT or overnight at 
4C, then washed, secondary for 1/2 hour at 37C).  And how do you store 
the slides once you have made them? Can they stay at RT or must they be 
frozen? Many thanks - Melissa


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