Stain Technic for Nissl's Granules Following Alcohol-Formol Fixation

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Abstract
1. Nerve tissue is fixed 2-4 hrs in a 5% solution of strong formalin in commerical 95% alcohol. 2. If dehydration is perfect, either chloroform or xylol may be used as a clearing agent. 3. A slow method of paraffin infiltration is advisable. 4. Sections should be cut 10-12 microns in thickness. 5. Coplin staining jars should be annealed by placing them on a rack in a pan of cold water, bringing the water to the boiling point, and allowing the jars to stand in boiling water for twenty minutes. 6. One per cent aqueous solutions of either methylene blue or Grübler's Neutral Roth are used as specific stains for Nissl's granules. 7. These stains are heated to boiling in a beaker, the slides are placed in the Coplin jars which are partially submerged in boiling water, and the hot stain poured into the jars. The flame beneath the water bath is turned down and the slides left for 20 minutes. 8. The excess of primary stain is washed off in 25% and 50% alcohol and the slides passed rapidly thru the alcohol series to absolute alcohol, and finally to xylol. 9. When counterstaining is desired, nigrosin in 1% aqueous solution, methyl orange, saturated solution in 50% alcohol, or, a 0.5% solution of eosin in 50% alcohol are recommended. These stains are used cold, and the slides are merely dipped in them after the excess of primary stain has been washed out in 25% and 50% alcohol. 10. If a cold primary stain is desired, a saturated solution of thionin in distilled water, acidified with 1% carbolic acid, will prove specific for the Nissl substance. Sections should be stained 5–10 minutes in thionin, then passed rapidly thru to absolute alcohol, and xylol. The same counterstains may be used as in the hot method. 11. Sections prepared by the hot method show little tendency to fade after ten years use. 12. Excepting neutral red, all the stains used in this technic are carried by the National Aniline and Chemical Company and are satisfactory. Coleman and Bell neutral red may be substituted for Grübler's Neutral Roth with good results.

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