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Both Protocol #1 and Protocol #2 are OK. However, we can use biochemical and histological work with protocol#1, whereas we can't use biochemical assay with protocol #2. Reason: After fixation with 4% PFA, tissues will be fixed, and western blot and other biochemicals, including qPCR, can't be done in the future.
Therefore, our Augusta site prefers protocol#1 in the light of future biochemical and histology work. If it needs only histology work, protocol #2 will be good.
Tissue banking protocols
Biochemical events and alterations should occur mostly during the first 7-14 days post-stroke, and thus might not be significant at 30 days post-stroke. The injured brain area may be significantly restructured and thus can be studied with the morphological and imaging analysis. If this is likely the case, we may store samples in the fixative vials at 4 degree or even room temp until use.
I also would like to suggest to collect died post-stroke animals into containers filled with a fixative solution as soon as we could, as the dead body may help us to understand the reason of animal death. For example, aged mice after stroke may die due to intestinal damage, sepsis, and systemic inflammatory response.
necropsy on drop outs due to death
Could you all please opine on whether you want to necropsy all dropouts due to death? Look for cerebral heme? Look for pneumonia? Urosepsis?