histology of cerebellum

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Shampa's picture
histology of cerebellum

i want to study the histology of cerebellum.Which staining procedure should i use? please provide me with the protocol for the same.

Monu's picture
Shampa, I would suggest you

Shampa, I would suggest you to take sections around 15 um. Then follow this protocol for staining. This protocol is for LFB. It stains the myelin and will be helpful to you.
Take to 95% alcohol. Stain in luxol fast blue solution - 2 hours at 60oC* or preferably overnight at 37oC. (Use a screw-topped plastic coplin jar.) Wash in 95% alcohol. Rinse in distilled. Start differentiation by agitating for 10 seconds in a jar of 0.005% lithium carbonate. This solution begins to loose its differentiating ability after a few uses so replace it often with fresh. Differentiate for 30-60 seconds in 70% alcohol. Rinse all slides in distilled water and check the staining with a microscope. Repeat the three steps above until grey and white matter are clearly distinguishable. Nuclei should be colourless and myelin should stand out turquoise on a very pale grey/blue background. Stain in 0.1% cresyl violet in 1% acetic acid - 10 minutes. Wash in distilled. Rinse in 70% alcohol. Differentiate in 70% alcohol until only nuclei and nissl substance are purple - approx. 30 seconds. Rinse quickly in 100% alcohol, clear and mount.
Other details:
Results will come like this:
Myelin - blue.
Red blood cells - turquoise.
Nuclei and nissl substance - purple.
* For the 2 hour timing use 0.05% lithium carbonate to differentiate instead of 0.005%, but be careful, it works much more quickly!
Luxol fast blue solution
Luxol fast blue MBS - 1g
95% alcohol - 1 litre
10% acetic acid - 5mls

Monu's picture
I am attaching a picture of a

I am attaching a picture of a control section for reference
I tried to put in jpg format directly but limit says 200kb only

Jitendra Sinha
Jitendra Sinha's picture
You can also go for H&E stain

You can also go for H&E stain but the best would be cresyl violet. If you go for IHC then try with Anti-reelin.

lidrus's picture
As suggested by Jiten, cresyl

As suggested by Jiten, cresyl violet is great over-arching (and simple) stain for detecting neurons and even sometimes glia (if you know structural features very well: www.pathology.vcu.edu/WirSelfInst/glialpath.html) within the cerebellum. With IHC, both neuronal types (Purkinje cells & granule cells) could be picked up with NeuN. They look so different from each other that that wouldn't be a problem to differentiate between the two population types. For the specific types of glia, there's the usual suspects - GFAP and/or vimentin (all glia), NG2 (oligo) etc. which are very popular stains and should be easily found with a quick lit search.
Good luck!

sencajal's picture
I think that staining

I think that staining procedure is totally depends upon your experiment? if you want to study the neuronal types and connections try golgi staining. There is no difference in cerebtu and cerebellum staining procedures all are same.