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Staining (March 2005)

Staining depends largely on the attachment of acid or basic dyes to proteins, and pH buffering, to acidify
or alkalize, can be done to ionize* tissue components (e.g., amino groups) so proteins behave as acidic or basic.
Neutral Red stains cell (Nissl) bodies; it more sharply differentiates gray & white matter than does cresyl violet.
Luxol Fast Blue stains the myelin sheath (phospholipids) of fibers. The mechanism of Luxol Blue is one of an
acid-base reaction with salt formation—the base of the lipoproteins in the sheath is replaced by the base of the
dye. Stainless steel electrode tips are marked by passing current through the electrodes after the perfusion to
deposit iron, stained BLUE in the tissue using Perls Iron Reaction, both during the perfusion (potassium
ferrocyanide, Bottle #3) and during staining (Ferrocyanide Hydrochloride). As a counterstain, neutral red
contrasts sharply with the blue iron. For experiments involving electrodes, avoid Luxol blue staining where the
recording was located since it could mask the blue mark of the electrodes. Sort slides into separate slide trays if
it is necessary to stain the fibers in other areas of the brain. DH# = dishes in fume hood; D## = dishes on
counter, numbered clockwise, upper left to upper right, then lower right to lower left, ending nearest fume

Staining for cell bodies only (Nissl staining with Neutral red) (for areas with electrodes)
DH1 Citri-solv 5 min (dewaxes thin sections if paraffin-embedded)
DH2 Citri-solv 5 min (defats thicker, frozen-sliced sections)
D01 100% alcohol 3 min (hydrates sections)
D02 95% alcohol 3 min (hydrates)
D06 Nanopure water 3 min
D10 Ferrocyanide HCl 10/30** min (stains ferric Fe3+ deposits Prussian blue in acidic sol’n)
D11 Nanopure water 3 min
D12 Acetate buffer 3 min (sharpens nuclear staining by acidic inhibition of non-nuclear staining)
D13 Nanopure water 3 min
D14 Neutral Red 20 min
D15 Nanopure water Rinse
D16 70% alcohol 2 min (Etoh series dehydrates & differentiates sections)
D17 95% alcohol 2 min
D18 100% alcohol 1 min (repeat D17/18 series if too dark; if too light repeat D01/02/11/12/13)
DH1 Citri-solv 5 min (clearing agent; removes dehydrant)
DH2 Citri-solv Until cover-slipping completed

Staining for cell bodies AND myelin (for tissue without electrodes) (Wijitha’s method):
D01 100% alcohol 3 min
D02 95% alcohol 3 min
D03 70% alcohol 1 hour
D04 Luxol blue overnight** in the oven (50-55 C, 16-18 h) (lid closed)
D05 95% alcohol 15 sec
D06 Nanopure water 3 min
D07 Lithium carbonate 3 min (alkalizing agent; begins to differentiate the tissue)
D08 70% alcohol 3 min (continues differentiation; if too dark, repeat D06-08)
D09 Nanopure water 5 min (if sections lifting off, lightly press with filter paper after adjusting the section)
D11 Nanopure water 5 min
D12 Acetate buffer, pH 4 3 min (re-acidify solution)
D13 Nanopure water rinse
D14 Neutral red 20 min (now’s time to move a 2nd slide tray from D04 to 05)
D15 Nanopure water rinse
D16 70% alcohol 2 min (Etoh series dehydrates & differentiates)
D17 95% alcohol 2 min
D18 100% alcohol 1 min
DH1 CitriSolv 5 min
DH2 CitriSolv Until cover-slipping completed
Dyes are generally ionizing compounds, and the actual coloring component is located in one or more of the
ions. Note that the terms used (acid, basic, neutral) bear no relationship to the pH of a solution of a particular
dye. Acid dyes have the coloring component in the anion (-), Basic dyes have the coloring component in the
cation (+), Neutral dyes have coloring components in both anion and cation, and Lysochromes do not ionize,
but color by dissolving into the target, usually a lipid. Regressive staining means that the tissue is deliberately
overstained and then destained (differentiated, remove excess stain) until reached proper endpoint. As long as
the slide is overstained, it doesn't matter whether it was in the staining dish for 1 or 10 min; differentiation also
removes dyes from the gelatin adhesive, producing a clear, transparent background. Differentiation is done in
dilute acid (for Luxol blue, acid alcohol because of greater solubility in alcohol). Differentiation is stopped
immediately by simply washing slides in water. If too much stain has been removed, a few more seconds in the
stain will correct the problem, and the whole process can be repeated. If too little has been removed, a few
more dips in acid alcohol will produce the correct endpoint.
**Dip longer if brain not perfused with fixative Bottle #3 (Potassium Ferrocyanide)

0.1% Neutral Red: (store in refrigerator in amber bottle/bring to RT before use.)

Neutral Red 1g
Thymol (Preservative) 0.5 g
Hot nanopure water 50 ml
Combine Neutral Red and Thymol in mortar and grind with pestle. Add heated water to ground powder in
mortar and grind some more. Cover and store overnight. Next day, regrind and add 950 ml nanopure water.
Mix well, filter. Can be re-used several times.

0.1% Luxol fast blue G (or Solvent Blue 38): (1/10th concentration of what we previously used)
Luxol G (Sigma #S3382-Solvent Blue 38) 1g
95% alcohol 1000 ml
10% glacial acetic acid 1 ml
Soluble in alcohols & phospholipids. Note: Store in refrigerator in amber bottle; can be re-used several times.
Before you start, turn the oven on and put Luxol blue dish in there for about 1-2 hours *lid on*, to get the
temperature between 50 and 55 Celsius. Stir the Luxol Blue before you put the slides in.

Acetate Buffer:
Sodium acetate 14.4 g
Nanopure water 600 ml
Glacial acetic acid 0.8 ml
Thymol (preservative) 0.5 g
Note: Check pH and adjust to 4 for Luxol Blue staining. If it’s less than 4 then adjust with 1M NaOH solution,
if more than 4, then add more acetic acid.

Ferrocyanide Hydrochloride:
Nanopure water 784 ml
Conc. HCl 16 ml
10% potassium ferrocyanide 16 ml
Nanopure water 16 ml
Potassium hexacyanoferrate(II) trihydrate 1.6 g

0.1% Lithium Carbonate Solution: (dissolves slowly, vortex 15-20 min; don’t store – effectiveness decreases
with use)
Lithium carbonate 1.0 g 0.6 g
Nanopure water 1000 ml 600 ml (just enough to cover the slides)

Modified 6-3-09, GZ