I'm pretty sure they are mostly macrophages since after a 2 hour incubation in M199 they are stuck with pseudopodia. My slides just aren't pretty enough for me. I'm using Fisherbrand Wight-Giemsa Stain on rabbit cells from a peritoneal wash (days after drawing monocytes to the location with a peritoneal proteose peptone injection). But my concern is that at some point my counts are off because I can't distinguish between macrophages and other large multi-nucleated cells.
My method:
20uL of 10^6 Trypan blue visualized cells are spread, dried using a Bunsen burner, and then fixed with Methanol for 60s.
Slide is flooded with 1mL Wright-Giemsa Stain and incubated 2 minutes.
Add 1.5mL 1X PBS at pH 7. Gently tipped to mix, for 1 minute.
Stands for 2min.
Rinsed well with De-Ionized water.
Dried and visualized at 400X.
Does it have to air dry rather than use a flame?
I am just having a hard time with the outer membranes. Should I use a different method?
Any help is appreciated!
Dear Barbara!
Please You take a look at this technique, it is very similar to yours, but there are differences in exposure time.
https://iai.asm.org/content/iai/37/1/64.full.pdf
FIG. 1. Experimental protocol.
This is a common problem if the stain used is old, or not mixed properly if using after a long time. I would suggest, you use a fresh bottle of stain (if using ready-made) or just make a new stock. We prepare Giemsa stain and when used after 4-6 months, we usually don't get good staining. Give it a shot.
You Can use fresh 1:10 dilution of giemsa stain from main stock. After Dilution, keep the stain in dark for 45 minutes followed by rinse with distill water by diping the slides in to it threafter keep the slides at 37 degree celsius for 15 min so that no water remains on it. Then enumerate under microscope. Hopefully it works!
Thanks
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