(Final full mosaic image is an epifluorescent slide scanner image)
#PainResearch #Immunofluorescence #Rnascope #Neuroscience
2 months agochaimeetsrusk
So beautiful ! If I were to detect GFP -both mRNA and protein, how would I do that ? I would be using different opal dyes/fluorescent antibody ?
3 months agomorgankwright26
Is your 405 channel ok? I see left to right gradient in only that channel, I had the same issue a few months back and needed a leica tech to come out to get even intensity🫠
3 months agoaindipandy
This is my favourite technique!!! Always do the RNAscope+immuno on same samples!!!
3 months agoscience_glitter
@matthew._.w7 Hi, the original RNAscope v2 kit protocol called for a 15-minute fix and a 30-minute ProtIV treatment (the protocol version from 2019). When I tried it back then, my tissue was always over-digested, so I optimized the heck out of the assay back then. I tried different incubation times of Protease III and Protease IV. I settled with Protease III for 10 seconds and across the board, it has always worked for me on every fresh frozen tissue sample I have tried (rodent, human, nervous and non-nervous tissue samples). It works without Protease as well, although my signal was slightly better with the Protease III for 10-seconds which is why I picked it. I think some degree of permeabilization is necessary for probe accessibility. I think optimal fixation and protease time are critical steps here. The newer ACD protocol now says 30-minute fix with a 30-minute ProtIV treatment and specifies that if over-digestion is observed, to reduce the protease time and “in some cases of severe over-digestion, use RNAscope Protease III.”
3 months agomatthew._._.7
If I remember correctly, the protocol for fresh frozen tissue is Protease IV for 30min at RT. Why do you do Protease III for 10 seconds? Seems like a waste of time to even perform the step, since P-III is weaker than P-IV, and 10 seconds instead of 30 minutes.
3 months agoborris_aldonza
Learned that many labs do digital deconvolution in Arivis in cases of RNA FISH co-staining with proteins since the FISH signal can appear so dirty. But yours look great already.
3 months agoscience_glitter
🤯 What is even crazier is that this tissue sample is human trigeminal ganglion. Its an extemely challenging dissection because it requires removal of the brain as it sits at the base of the skull. We also dissected it 24 hours post mortem and the mRNA/protein and the morphology of the sample was still intact... I remember when I used to do animal dissections in the past, I was always so careful and would go as fast as possible but this just goes to show that tissues can really last a long time as long as they are kept cold. It really comes down to preparation and good handling
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