Staining Recipes

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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 for
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  Staining (March 2005) Staining depends largely on the attachment of acid or basic dyes to proteins, and pH buffering, to acidifyor 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 anacid-base reaction with salt formation—the base of the lipoproteins in the sheath is replaced by the base of thedye. Stainless steel electrode tips are marked by passing current through the electrodes after the perfusion todeposit iron, stained BLUE in the tissue using Perls Iron Reaction, both during the perfusion (potassiumferrocyanide, Bottle #3) and during staining (Ferrocyanide Hydrochloride). As a counterstain, neutral redcontrasts sharply with the blue iron. For experiments involving electrodes, avoid Luxol blue staining where therecording 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 oncounter, numbered clockwise, upper left to upper right, then lower right to lower left, ending nearest fumehood. Staining for cell bodies only (Nissl staining with Neutral red) (for areas with electrodes) DH1Citri-solv5 min (dewaxes thin sections if paraffin-embedded)DH2Citri-solv5 min (defats thicker, frozen-sliced sections)D01100% alcohol3 min (hydrates sections)D0295% alcohol3 min(hydrates)D06Nanopure water3 minD10 Ferrocyanide HCl10/30** min (stains ferric Fe 3+ deposits Prussian blue in acidic sol’n)D11Nanopure water3 minD12Acetate buffer3 min (sharpens nuclear staining by acidic inhibition of non-nuclear staining)D13Nanopure water3 minD14 Neutral Red20 min D15Nanopure waterRinseD1670% alcohol2 min (Etoh series dehydrates & differentiates sections)D1795% alcohol2 minD18100% alcohol1 min (repeat D17/18 series if too dark; if too light repeat D01/02/11/12/13)DH1Citri-solv5 min (clearing agent; removes dehydrant)DH2Citri-solvUntil cover-slipping completed Staining for cell bodies AND myelin (for tissue without electrodes) (Wijitha’s method): D01100% alcohol3 minD0295% alcohol3 minD0370% alcohol1 hour D04 Luxol blueovernight ** in the oven (50-55 C, 16-18 h) (lid closed)D0595% alcohol15 secD06Nanopure water3 minD07Lithium carbonate3 min (alkalizing agent; begins to differentiate the tissue)D0870% alcohol3 min (continues differentiation; if too dark, repeat D06-08)D09Nanopure water5 min ( if sections lifting off, lightly press with filter paper after adjusting the section )D11Nanopure water5 minD12Acetate buffer, pH 43 min (re-acidify solution)D13Nanopure waterrinseD14 Neutral red20 min (now’s time to move a 2 nd slide tray from D04 to 05)D15Nanopure waterrinseD1670% alcohol2 min (Etoh series dehydrates & differentiates)D1795% alcohol2 minD18100% alcohol1 minDH1CitriSolv5 min  DH2CitriSolvUntil cover-slipping completed * Dyes are generally ionizing compounds, and the actual coloring component is located in one or more of theions. 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 thecation (+), 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 deliberatelyoverstained and then destained ( differentiated  , remove excess stain) until reached proper endpoint. As long asthe slide is overstained, it doesn't matter whether it was in the staining dish for 1 or 10 min; differentiation alsoremoves dyes from the gelatin adhesive, producing a clear, transparent background. Differentiation is done indilute acid (for Luxol blue, acid alcohol because of greater solubility in alcohol). Differentiation is stoppedimmediately by simply washing slides in water. If too much stain has been removed, a few more seconds in thestain will correct the problem, and the whole process can be repeated. If too little has been removed, a fewmore 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 Red1 gThymol (Preservative)0.5 gHot nanopure water50 mlCombine Neutral Red and Thymol in mortar and grind with pestle. Add heated water to ground powder inmortar 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)1 g95% alcohol1000 ml10% glacial acetic acid1 mlSoluble 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 thetemperature between 50 and 55 Celsius. Stir the Luxol Blue before you put the slides in. Acetate Buffer: Sodium acetate14.4 g Nanopure water600 mlGlacial acetic acid0.8 mlThymol (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 water784 mlConc. HCl16 ml10% potassium ferrocyanide16 ml Nanopure water16 mlPotassium hexacyanoferrate(II) trihydrate1.6 g 0.1% Lithium Carbonate Solution: (dissolves slowly, vortex 15-20 min; don’t store – effectiveness decreaseswith use)Lithium carbonate 1.0 g0.6 g Nanopure water1000 ml600 ml (  just enough to cover the slides) Modified 6-3-09, GZ
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