Hi, I will soon be preparing frozen mouse lung tissue for immunohistochemistry. I hear that with formalin based fixation, immunohistochemistry would be difficult to perform. I have also read protocols which inflate the lungs with a 50% OCT/50% PBS mixture, then freeze the lungs in OCT. Can anyone with experience with lung preparation for IHC share some thoughts on this?
Thanks!
Hello
Yes this true should be inflated lungs before freezing: Make 1:1 OCT: PBS .
Follow the protocol here :
http://mousepheno.ucsd.edu/Protocols.shtml
for IHC see this link :
https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-frozen-tissue-sections
Good Luck
We rather succesfull work with the Karnua solution (ethanol 96% -6 parts: hlorophorm –3 parts: acetic acid –1 part),-2 hours and then carry to the 70% ethanol, with the following keeping.
Follow the protocol here
https://www.rndsystems.com/protocol-types/immunohistochemistry-ihc-protocols
Unfixed frozen sections are preferred, if IHC on formalin-fixed tissue is difficult or impossible for some antigens. It is also preferred, if you perform IF because of autofluorescence of fixed tissue.
Fixed tissue is preferred to get better morphology. But if you already fix the tissue, the questions comes up, why you don't perform paraffin-histology at all.
I have used the 1:1 OCT/PBS and also, just OCT warmed up until it is less viscous and inflate the lungs. If you want to use a fixative. Mix 1:1 8% Paraformaldehye with OCT and use that to inflate, then place the lung in 4% Paraformaldehyde at 4C overnight, cryoprotect in 30% sucrose at least 24 hours at 4C and freeze in OCT. Both methods reduce autofluorescence and provide good morphology.
Joan T Zary Oswald do you first try to deflate the lung, before inflating using the OCT/PBS mixture? How do you avoid bubbles? While I have done a lot of embedding, sectioning, IHC, a new challenge of trying to detect activated macrophages in mouse lung tissue is on my plate. Thank you for your advice! It may be that paraffin is, in fact, better. I have worked mainly with fixed tissues in acrylamide, sucrose/oct, or OCT. I have little experience with paraffin processing. Above, when you state the paraformaldehyde/OCT mixture inflation, do you somehow tie off or close the trachea to keep the lung inflated before placing in 4% pfa overnight? I'm sorry if these questions are silly - trying to get as much info as possible! Thanks so much!
The lung deflates a little on its own as you are getting ready to inflate with OCT, PFA or OCT/PFA mixture. I haven't had a problem with bubbles. Yes, do tie off the trachea with suture. There will be some leakage, but enough stays to preserve the morphology.
Joan T Zary Oswald thank you kindly for your guidance. It is so valuable. As I think through the steps of this procedure, I have a couple more questions :)
I assume you are not perfusing before the inflation with OCT/PFA?
Approx. volume of OCT/PFA? (should I be worried about blowing out the lung alveoli if we use too much volume? )
Finally, when you embed in OCT, at this point have you dissected out a piece of a lung and what is the most common plane or even area of the lung that is sampled for a routine H & E analysis of overall lung structure?
I apologize for all the questions, we are very grateful for your expertise!
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