The problem you are having is most likely related to the fact that you are trying to stain 10 µm sections. With sections so thin 1) there aren't enough neurons to show regional boundaries very clearly, 2) for the same reason staining is usually very light even under the best of conditions and 3) loss of some Nissl substance is inevitable because the cell membrane of virtually every neuron has been cut. To preserve the contents of the cell stronger fixation is generally required.

To optimize what staining you can get, I have attached is a simple protocol (from Paul (2008) CSH protocols). This protocol is fairly typical and should work as is, but can be improved with the following modifications:

2x2 min for each alcohol step,

add a differentiation step (1% glacial acetic acid in 95% ETOH),

use Xylene (3x15 min) instead of Hemo-De for clearing, and

use DPX mounting media instead of Permount.

There are only a few basic problems one can have with Nissl staining: overstaining, understaining, poor differentiation or poor clearing.

If the tissue is over stained, use the differentiation step (above) to remove excess stain.

If the tissue is under stained, re-stain it—simply reverse the protocol at any point to rehydrate the tissue before re-staining.

Poorly differentiated tissue is generally either incomplete clearing of the stain from myelinated areas or overly light Nissl staining of the cells. For the first, the GAA differentiation removes stain from the interstitial spaces faster than the cellular staining. However, it is not difficult to reduce overall staining, producing the later, overly-light Nissl, which can be re-stained as needed.

Poor clearing results from either insufficient time or contamination in the clearing step. It is necessary to take the tissue through a series of clearing, or de-fatting/de-myelination, steps before coverslipping. These steps are usually Xylene, or some less-toxic alternative like Hemo-D. Problems here are usually due to insufficient dehydration of the tissue. Xylene has very little tolerance for water (Hemo-D has even less) and can precipitate and/or discolor the tissue. Avoid these issues w/multiple 100% ETOH steps prior, multiple clearing steps, and replacing the solutions regularly.

Clearing also takes time. While 15 min in xylene is adequate for Nissls, other stains/reactions may require longer. Also note that while xylene alternatives are capable of clearing to the same degree, as a rule they take 3-4X longer to do so.

Your question is not clear enough whether you wish to know how to prepare the CV solution or how to perform the CV staining with slide glass containing tissue. So please make the clear statement I can provide the protocol that I am using it currently.

Dear Bhakta Prasad Gaire, Thank you very much for your prompt response. I wanted to stain brain tissue (10 micron slice) with CV, but I failed. I bought Cresyl violet (acetate) for microscopy Certistain® from (http://www.merckmillipore.com/india/chemicals/cresyl-violet-acetate/MDA_CHEM-105235/p_zyOb.s1LZb0AAAEWZuEfVhTl). It would be great if you please share your protocol both 1. CV solution preparation and 2. Staining brain tissue.

Dear Bhakta Prasad Gaire.

Thanks once again. Have a nice day.

Dear Bhakta Prasad, why do you use chloroform? I'm interested in the chemical reasons. Is it just for deparaffination or an additional reason, because you call it "blocking"?

Chloroform ethanol mixture is used just for another fixative solution. That increase the strength of staining. if you don't wish you can skip this step. I have done both...

Hello Bhakta..i have done the cresyl violet staining several times and luckily found the good results...here i am sending you the preparation and staining protocol for cresyl violet stain..I'm sure it will definitely work.!!

All the best.

Protocol for CV stain preparation

Staining protocol..!

Dear Aarti Nagayach. Many thanks for the protocol.

The problem you are having is most likely related to the fact that you are trying to stain 10 µm sections. With sections so thin 1) there aren't enough neurons to show regional boundaries very clearly, 2) for the same reason staining is usually very light even under the best of conditions and 3) loss of some Nissl substance is inevitable because the cell membrane of virtually every neuron has been cut. To preserve the contents of the cell stronger fixation is generally required.

To optimize what staining you can get, I have attached is a simple protocol (from Paul (2008) CSH protocols). This protocol is fairly typical and should work as is, but can be improved with the following modifications:

2x2 min for each alcohol step,

add a differentiation step (1% glacial acetic acid in 95% ETOH),

use Xylene (3x15 min) instead of Hemo-De for clearing, and

use DPX mounting media instead of Permount.

There are only a few basic problems one can have with Nissl staining: overstaining, understaining, poor differentiation or poor clearing.

If the tissue is over stained, use the differentiation step (above) to remove excess stain.

If the tissue is under stained, re-stain it—simply reverse the protocol at any point to rehydrate the tissue before re-staining.

Poorly differentiated tissue is generally either incomplete clearing of the stain from myelinated areas or overly light Nissl staining of the cells. For the first, the GAA differentiation removes stain from the interstitial spaces faster than the cellular staining. However, it is not difficult to reduce overall staining, producing the later, overly-light Nissl, which can be re-stained as needed.

Poor clearing results from either insufficient time or contamination in the clearing step. It is necessary to take the tissue through a series of clearing, or de-fatting/de-myelination, steps before coverslipping. These steps are usually Xylene, or some less-toxic alternative like Hemo-D. Problems here are usually due to insufficient dehydration of the tissue. Xylene has very little tolerance for water (Hemo-D has even less) and can precipitate and/or discolor the tissue. Avoid these issues w/multiple 100% ETOH steps prior, multiple clearing steps, and replacing the solutions regularly.

Clearing also takes time. While 15 min in xylene is adequate for Nissls, other stains/reactions may require longer. Also note that while xylene alternatives are capable of clearing to the same degree, as a rule they take 3-4X longer to do so.