I just planned study with rat lungs. I had a good result with formaldehyde fixation. I recently notice paraformaldehyde (in PBS) used for lung fixation by researchers and they generally arranged fixation time 4 hour. I wonder can lung tissue wait in PBS about 1 week after 4 hour PFA fixation. and then I start tissue processing.
Dear Yasin,
as long as AND at least you don't fix the rat lungs initially by state-of-the-art perfusion fixation I guess that "fixation" results after 4 hours( @ 4°C?, @RT?) penetration time (naturally depending a bit also on the specimen's size) are much too short (for a "perfect fixation" in state-of-the-art and 'chemical terms', see /cf Kiernan paper on:
John A. Kiernan: "Formaldehyde, formalin, paraformaldehyde and glutaraldehyde:
What they are and what they do."
Article published in Microscopy Today 00-1 pp. 8-12 (2000). [Copyright (c) 2000 by Today Enterprises. Reproduced with permission.
Cf. @ http://publish.uwo.ca/~jkiernan/formglut.htm
For histology and/or IHC I would recommend at least [buffered FA/NBF-] fixation for 4-5 h at RT, followed by at least fixation over night [usually then done at 4°C....traditionally (:-)) ], then specs are removed from the fridge - warming to RT for further 1-2 hrs (best using always specimen rotator) - follow recommendations according to Kiernan! - up to 2-3 days, in the refrigerator also 1-2 days longer. When correct fixation then has been achieved or can be expected to be finished (always must meet your examination task!, i.e. Histology-Morphology, IHC, ICC, RNA-DNA matters, IF, CLSM....,etc,. etc., 1-3x washing in PHOSPHATE BUFFER solution (pH 7.2 - or 7.3 - or 7.4), also whole washing procedure(s) can last up to 2 days (rec.@°4C), then you easily and cheerfully can start the tissue processing (guessing into paraffin...). So called "overfixation" is - for sure - not to be expected, except you have to deal with a tricky, delicate Immuno-biomolecular task.
If you intend to use specimens for classical histochemistry or IHC, you could add still in the second buffer washing solution e. g. + 50 mM NH4Cl [Ammoniumchloride) for blocking free aldehyde groups (as well as quenching autoflurorescence -if present and otherwise troubling). Good luck and best wishes, W. M.
Dear Amal Hassan
, thank you for posting! Just for the convenience of other Readers I‘d like to add the citing reference:==> Hausmann R. (2006) Methods of Lung Fixation. In: Tsokos M. (eds) Forensic Pathology Reviews. Forensic Pathology Reviews, vol 4. Humana Press. https://doi.org/10.1007/978-1-59259-921-9_15
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