July 15, 2016

Tissue Staining



The paraffin is removed from the section with xylene. If the tissues are to be stained with an aqueuous solution then the slides must rehydrated in graded ethanol baths. Unless a time is indicated the approach is to gently agitate the slides by repeated immersion ~20x in each bath:


  1. Xylene for 2 min. (x3 changes)
  2. 100% EtOH (x2)
  3. 95% EtOH (x1)
  4. 80% EtOH (x1)
  5. H2O (x1)

Hematoxylin and Eosin (H&E)

This is the most conventional stain for formalin fixed paraffin sections. The hematoxylin stains negatively charged nucleic acids (nuclei and ribosomes) blue. The eosin stains proteins pink. The hematoxylin or the eosin can also be used by themselves in more dilute form as counterstains for immunoperoxidase staining. To do this dilute the stain 1:4 with H2O or EtOH, respectively. Slides to be stained in must be washed in ethanol first as listed below for the conventional protocol:


  1. Harris hematoxylin: 1X stock. Anatech Lmtd.(616) 964-6450
  2. Eosin: 1X stock. Cat #837.
  3. Acid alcohol: 76.6% EtOH, 1/300 v/v conc. HCl. (230 mL EtOH, 70 mL H2O, 1 mL HCl)
  4. Ammonia Solution: 0.084 w/v NH4OH (1 L H2O 3 mL 28% w/v NH4OH stock).


  1. Hematoxylin, 2 minutes (x1)<>
  2. Running water (x1)
  3. Acid alcohol (x1)
  4. H2O (x1)
  5. Ammonia solution (x1)
  6. Running water 5 minutes (x1)
  7. 80% EtOH (x1)
  8. Eosin 15 seconds
  9. 95% EtOH (x2)
  10. 100% EtOH (x2)

Wright Giemsa

This is the conventional stain for blood smears and bone marrow cytology. It is usually performed on an automated slide stainer (see pathology or Bernstein lab).

Methyl Green

(2% (w/v) methyl green in 0.1 M NaOAc, pH 4.2)

Methyl green is a nuclear counter stain which works nicely for immunoperoxidase stained slides. It is difficult to control the intensity of the stain however since it washes out in both aqueous and organic solutions and this will depend on how quickly you mount the slides. Mix 918 mL of 0.1N acetic acid with 331 mL of 0.1 M NaOAc and adjust pH to 4.2 with NaOH. Add 25 gm of methyl green dye. Filter through Whatman #2 filter paper.


  1. H2O x 10-15 sec.
  2. Methyl green x 5 min.
  3. H2O (x2).
  4. Air dry.

New Methylene Blue

This stain is useful for distinguishing reticulocytes from mature RBCs in the peripheral blood. Mix whole blood 1:1 (v:v) with New Methlylene Blue (Ricca Chemical Co., Arlington Heights, IL). Incubate 10 minutes. Count on hemocytometer.

Benzidine Stain

This is a specialized stain which identifies erythroid cells (RBCs and their precursors). It gives them a golden brown color.


  1. Methanol, 10-15 seconds.
  2. Benzidine, 5 min: [1% w/v of 3,3' dimethoxybenzidine in methanol] (This is toxic stuff).
  3. Peroxide 2.5 min. (1 vol 30% H2O2 plus 11 vol. 70% EtOH)
  4. Dionized water wash, 2.5 min.
  5. Hematoxylin stain, 1.5 min.
  6. Rinse in tap water, 8 min.
  7. Air dry.

Acetylcholine esterase (AChE) Stain


  1. Fix in 5% glutaraldehyde x 15 min.
  2. H2O (x3)
  3. Flood slide with fresh AChE stain. (5 mM NaOAc, pH5; 1mM glycine; 0.2 mM CuSO4; 1.15 mg/ml acetylthiocholine iodide: Sigma A5751)
  4. Incubate in petri dish o.n.
  5. Rinse in H2O (x3), air dry.

Tags: Fero-Histology, Fero-Lab-Protocols