Tissue Staining
Deparafinization: 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:
Xylene for 2 min. (x3 changes)
100% EtOH (x2)
95% EtOH (x1)
80% EtOH (x1)
H2O (x1).
Alternative method……..
Immunostaining
In Plastic Tubs (200 mL): Deparafinize:
1. Xylene 2' (x3)
2. 100% EtOH 2' (x3)
3. 70% EtOH 2' (x1)
4. PBS 5' (x3).
De-Paraffinization of Tissue Sections
In order to proceed with sample preparation, paraffin must be removed from tissue
sections.
Materials
Xylene
Ethanol
Distilled Water
Copplin Jars or other solvent containers
Procedure
1. Section paraffin blocks at 5-10 microns. 5 microns is optimal for LCM, but the thickness
should be dependant on the cell (nuclei) diameter that is being worked on.
2. Place ribbons into a water bath at 42° to smooth out and eliminate folds and wrinkles.
3. Mount sections onto glass slides. Air dry overnight at room temperature. If sections do
not adehere to the slide sufficiently they can be baked at 42 C for up to 8 h.
4. Dip the slide containing the tissue section in the following solutions for the specified
times.
Xylene
5 minutes
Xylene
5 minutes
100%Ethanol
30 seconds
95% Ethanol
30 seconds
70% Ethanol
30 seconds
Distilled Water
30 seconds
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 eosin must be washed in ethanol first as
listed below for the conventional protocol:
Reagents:
Harris hematoxylin: 1X stock. Anatech Lmtd. Cat #842. (616) 964-6450
Eosin: 1x stock. Cat #837.
Acid alcohol: 76.6% EtOH, 1/300 (v/v) conc. HCl. (230 mL EtOH, 70 mL H2O, 1 mL HCl)
Ammonia Solution: 0.084 (w/v) NH4OH (1 L H2O 3 mL 28% w/v NH4OH stock).
Procedure:
Hematoxylin, 2 minutes (x1)
Running water (x1)
Acid alcohol (x1)
H2O (x1)
Ammonia solution (x1)
Running water 5 minutes (x1)
80% EtOH (x1)
Eosin 15 seconds
95% EtOH (x2100% 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 .
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.
H2O x 10-15 sec.
Methyl green x 5 min.
H2O (x2).
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.
Methanol, 10-15 seconds.
Benzidine, 5 min: [1% w/v of 3,3' dimethoxybenzidine in methanol] (This is toxic stuff).
Peroxide 2.5 min. (1 vol 30% H2O2 plus 11 vol. 70% EtOH)
Dionized water wash, 2.5 min.
Hematoxylin stain, 1.5 min.
Rinse in tap water, 8 min.
Air dry.
Acetylcholine esterase (AChE) Stain
Fix in 5% glutaraldehyde x 15 min.
H2O (x3)
Flood slide with fresh AChE stain. (5 mM NaOAc, pH5; 1mM glycine; 0.2 mM CuSO4; 1.15 mg/ml
acetylthiocholine iodide: Sigma A5751)
Incubate in petri dish o.n.
Rinse in H2O (x3), air dry.