Edited by Bob Price University of South Carolina School of Medicine

Selected postings are from discussion threads included in the

Microscopy ( and Confocal Micros- copy ( listservers from September 1, 2019 to October 31, 2019. Postings may have been edited to conserve space or for clarity. Complete listings and subscription information can be found at the above websites.

Techniques and Problem Solving

Confocal Microscopy Listserver Problems with old samples and immunohistochemistry I need some help troubleshooting an immunohistochemisty

(IHC) protocol for a lab. Ninety nine percent of what reaches my facility is fresh, or relatively fresh material from tissue cul- ture or small animal experiments. Te samples we are having issues with are from human brains that have been in forma- lin for a number of years (some for over a decade or two) and, according to records, the tissues were not prepared immediately, rather some were processed 48 hours post-mortem. Te IHC has seen little success: we are getting mostly background with little specificity. A couple of samples do seem to have worked, but we cannot rely on one or two slides (out of many dozens). In some samples, even the DAPI is not working well. Here are some things that were tried by the lab in order to improve the IHC protocol:

• 24 h incubation in paraffin (as part of the paraffin block protocol).

• Antigen retrieval with citraconic-anhydride (supposed to handle the formalin crossover).

• Increased primary antibody concentration to 1:100. • Tyramide

staining in order to strengthen the signal

• Two blocking protocols were tried, both of which are supposed to work in this type of IHC: CAS-Block™ and Goat serum

• A MaxBlock™ kit was used to lower the high autofluorescence inherent to these samples. Can this be blocking the antibodies? We have never worked with this before and it gives a black tint to all the samples. Supposedly without this, the samples are impossible to image.

Does anyone have experience working with such old samples and have successfully done IHC with multiple colors? Any tips or ideas are very much welcome. Avi Jacob

Human brain and postmortem: Tis is definitely a tough

one. I would first try to reduce the autofluorescence without staining the sample. For example you can try bleaching the

42 doi:10.1017/S1551929519001172 acid Signal Amplification (TSA) for secondary

sample (e.g., exposing the sections overnight in a hood with a UV lamp). NAD and flavins usually bleach very nicely. You can also try hydrogen peroxide. Once you find a way to reduce the autofluorescence you can optimize the staining. I would guess that paraffin embedding would make things worse but if you need it, from what we have seen Sudan Black seems to reduce the fluorescence due to paraffin embedding. However it sounds like MaxBlock™ is similar. You could also try to use linear unmixing on the microscope. Sometimes you cannot see the signal, but if you manage to exclude the autofluorescence spectrum you find that the staining has worked. Ten it is easier to troubleshoot. You can acquire the ‘pure’ fluorophore spectrum by immunostaining cells in culture. Ten you use this spectrum to unmix your real sample. Good luck! Sylvie Le Guyader

With brain samples prepared under much more favorable

conditions I have had success spectrally unmixing the autoflu- orescence using a 32-PMT Nikon A1 (no commercial interest). Leica and Zeiss scopes should have comparable functionality though Leica uses a different mechanism. Separately, if you can find a FLIM system then you may be able to isolate the antibody signal in the frequency domain, especially if you use probes with really distinct lifetimes. Lanthanides would be ideal, but I’ve never seen those used for imaging, just plate reader FRET- FLIM. Long-shiſt probes with built in FRET might also do. It’s a technical challenge but your particular needs may justify the trip and effort. Timothy Feinstein

Microscopy Listserver

HM20 resin issues – HELP! Has anyone had issues with infiltration and embedding of liver ) with HM20 resin for immunoelectron microscopy?

(less than 1 mm2

I’ve been having issues with chatter and with areas in the tissue not infiltrating well. I first tried an old batch of HM20 resin, and then a new batch of HM20 resin. Both ended up with white, crusty tissue embedded in the resin (mostly the top and middle part of the tissue). Aſter milling until all of the crusty part was gone the tissue seemed okay to section, but there was too much chatter. Tere was no improvement aſter further curing at -20°C with UV light for 24 hours or more. Rudy Alvarado

I think the infiltration is incomplete due to incomplete dehy-

dration. I would suggest increasing the time for dehydration in alcohol to enhance infiltration. If you share your embedding pro- tocol I can suggest further steps. Regan M.

To answer your questions, this is what I have done:

Trial 1 - room temperature - immunoelectron microscopy pro- cessing protocol: Liver pieces were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in PBS (pH: 7.20). Tey were washed in PBS, water, and dehydrated in a short graded ethanol series (25, • 2020 January

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