Feb 03, 2025
Nissl Staining with Thionin
- 1University of Virginia
Protocol Citation: Tracy Larson 2025. Nissl Staining with Thionin. protocols.io https://dx.doi.org/10.17504/protocols.io.261ger8edl47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 02, 2025
Last Modified: February 03, 2025
Protocol Integer ID: 119454
Abstract
A "Nissl stain with thionin" is a histological technique used to visualize the cell bodies of neurons in tissue sections, where the primary dye, thionin, binds to the negatively charged components of Nissl bodies (clusters of rough endoplasmic reticulum) within the neuronal cytoplasm. This selective staining allows for clear identification of neurons and their distribution within the tissue. The method is particularly useful for studying neuronal morphology, cell density, and the cytoarchitecture of various brain regions. During the staining process, tissue sections are exposed to a thionin solution, which stains the Nissl bodies, and the excess stain is often removed through a differentiating solution to enhance contrast. Under the microscope, neurons appear as distinct blue or purple structures, making it easy to distinguish them from non-neuronal tissue.
Materials
4% PFA
Paraformaldehyde 4.0g
1x PBS 100ml
*heat to get PFA into solution, then cool to 4C before use
**don’t boil!!
10xPBS, phosphate buffered saline
To make 1 L of 10X stock:
NaCl 80.0 g
KCl 2.0 g
Na2HPO4 14.4 g
KH2PO4 2.4 g
nano H20 to 1.0 L
*dilute to 1X working concentration
Thionin Stock
Stock 1.3% thionin:
13 gm Thionin
1000 ml distilled H2O
*Stir and heat gently for 1 hour. Filter the solution after the dye has dissolved. Store in a stoppered bottle. Use high purity thionin, such as Sigma T3387.
Thionin Buffer
1 M Acetic Acid (HAc):
58.5 ml glacial acetic acid
dilute to 1 liter with distilled water
2. 1 M Sodium Hydroxide (NaOH):
50 gm sodium hydroxide pellets
dissolve to 1 liter with distilled water
*Mix buffer reagents together and adjust pH before mixing with the thionin stock in the formulas below.
Thionin Stain
80.0 ml 1M HAc
14.4 ml 1M NaOH
305.6 ml Thionin stock
Tissue Fixation
Tissue Fixation
Fix tissue in 4% PFA for 15 min
Rinse in 1x PBS at least once for 5 min
Staining
Staining
Rinse in dH2O for 3-5 min
Stain in Thionin stain for 2-7 min, depending on number of times stain has previously been used
* Typical schedule: 1-3 uses = 2-3 min; 4-6 uses = 3-4 min; 6-10 uses = 5-7 min
**Re-make solution after 10 uses
Dehydrate:
70% EtOH with a few drops of Acetic Acid, 15-30 sec, dip twice
70% EtOH, 15-30 sec, dip twice
95% EtOH, 30 sec to several minutes
*This step differentiates the nuclei stain (purple) from the cytoplasm stain (blue)
**Watch for desired “darkness” of stain (ideally medium blue with a slight hue of
purple)
100% EtOH, 30 sec with several swishes back and forth
*Important to completely dehydrate/remove all water from the sections before
moving to xylenes
Mounting Coverslips
Mounting Coverslips
Clear in xylenes for 3-5 min
*Look for clearing of “fuzzy” or “drippy” (this is moisture trapped in the tissue)
appearance before mounting
Drop a dime-seized drop of DPX onto each slide and gently place a 24mmx60mm coverslip on top