[Histonet] Histonet Digest, Vol 163, Issue 15 Research histology and animal IHC

[Steve McClain]

Underfixation being the greater problem, for animal research I tell everyone 24 hours fixation. After 24 hours in clean formalin, Stop fixation by pouring off and transfer to 50% reagent alcohol. Using standard fixation and slow processing, repeatable near-perfect histology and IHC can be made w comparable staining in small biopsy and large samples alike. 

There is no right answer but kindly choose one method and teach that in your lab, better consistent than correct. IHC is antibody dependent and species dependent, and fixation dependent but IHC  is especially section dependent. Quite predictably excellent slides and excellent IHC can be made from tissues in almost every species given 24 hours fixation and overnight processing, the methods we first learned in school 30-40 years ago.

For 2mm samples in human clinical testing using common antibodies, we can acceptably shorten this to minimum 8-12 hours fixation and rapid 2 hour processing using fresh reagents w a wee bit of heat. But in research what is gained by it? Underfixed tissue causes more spurious results than over fixation. 

In weird species, having weird collagens weird cells, weird antibodies and weird questions all confounding the microscopist who needs spurious results?

In research studies w IHC endpoints  and morphometric work, we found results improved by stopping fixation at 24 hours, transferring the whole batch to 50% alcohol and then slow 12H to 20H processing wo heat.  For really large slices processed in teabags or even toenails, an extended 16-20H processing can be useful. Adding more time in paraffin and xylene to the 12 hour.  Counterintuitive in the era of rapid processing schedules, but slow processing produces consistent IHC results between small biopsy and large samples.

Research processing for science
RT 12 hours fresh reagents NO heat. NO formalin. We run ALL tissue processors wo formalin (station 1 is 50% alcohol). Long processing wo heat or prolonged 50% alcohol storage for days or weeks at RT does not appreciably harden tissue.  

Removing formalin completely from the tissue processors was done gradually, first on the animal research machines and rapid processing machines. Eventually the last VIP5 w the overnight run is where I met w resistance from my histotechs. Even though we had shown no issues w research samples or when short processing human samples, they wanted formalin on the processor- in case. 

Quite logically once fixed tissues are in 50% alcohol additional processing time in formalin serves no purpose.  My histotechs belatedly gave in after we ran the processors for 6 months with only 1 minute in the formalin station 1. Our medical director covertly reprogrammed formalin station 1 to one minute and added back time to the alcohol in station 2 wo their knowledge (Choose your battles.)

Steve A. McClain, MD
McClain Laboratories, LLC

On Jun 16, 2017, at 13:20, "histonet-request at lists.utsouthwestern.edu" <histonet-request at lists.utsouthwestern.edu> wrote:

>> Can anyone tell me what, if any, guidelines there are for fixation time for
>> animal tissue with potential subsequent IHC stains.  I am very familiar
>> with CAP guidelines for receptor testing with breast cases, but I can't
>> seem to find much on IHC for animal tissue.
>> 
>> Thanks,
>> Cristi