Hello
I am trying to perform a blood analysis of micronucleus of lymphocytes in mice peripheral blood. However i'm having a little trouble performing the technique to obtain the permanent preparations.
Any suggestions on how to prepare, stain and preserve the blood?
Best Regards
I am not sure what kind of staining protocol you are looking for, but here is a general guide to making, fixing and staining blood smears
http://www.slideshare.net/ankur16491/blood-smear-staining
Try and get your hands on a 'diff-quick' or 'quick-dip' dye set (http://www.ihcworld.com/_protocols/special_stains/diff_quick_ellis.htm). Most histopathology providers stock it, I think ours is from Fronine, called Quick Dip I and Quick Dip II (http://www.fronine.com.au/haematology-stains.htm). You'll also need 100% methanol, or you can use their 'Quick Dip Fixative'.
- Smear a drop of blood onto a glass slide (see Nicole's link)
- Air dry (must be properly dry)
- Submerge in methanol, 20 seconds
- Submerge in Quick Dip I (red), 20 seconds, blot off excess
- Submerge in Quick Dip II (dark blue), 20 seconds
- Rinse with tap water
- Air dry
- Using a glass (NOT PLASTIC or it will melt) pipette/dropper/rod, place 1 drop of DPX mounting media on slide (http://www.sigmaaldrich.com/catalog/product/sigma/06522?lang=en®ion=AU). Make sure to the read the MSDS about storage and handling
- Place coverslip on top, leave for 1-2 hr or overnight to dry
Now you have a preserved stained blood slide.
If you are interesting only in lymphocytes ; it is better to separate the mononuclear cells layer by using lympholyte solution (Lympholyte®
Cell Separation Media)and centrifugation, after transfering that layer in another tube, reconstitute with buffer saline; fix with absolute methanol, and stain with diff quick or may grunwald giemsa stain.
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