Do not cut the brain until it has been fixed. After paraffin embedding the brain should be cut with a scalpel and embedded cut surface down. In this way you can decide whether you want to view coronal or sagital sections.

Pituitary: After the brain is removed the pituitary will generally still be sitting on the floor of the skull between the two large cranial (trigeminal) nerves that run longitudinally along the bone. The pituitary will be a 2x4 mm strip of pale tissue running crosswise between these two nerves. Under the dissecting microscope the thin meningeal membrane that overlies the pituitary should be torn away with forceps. Gently lift out the pituitary with the tip of a curved 26G needle or with fine forceps. Wrap it in a small piece of lens paper and fix it in cassettes. All 3 tissue layers will be visualized if it is embedded upside (posterior) side down.
Tissue Processing and Sectioning

Decalcification of bone:

  After fixation bone must be decalcified or else it won't cut on the microtome:
  11% formic acid with a stir bar overnight in a fume hood.
  Rinse in running water for 30- 60 minutes (the smell should be gone).

70% Ethanol

After adequate fixation tissues are transferred to 70% ethanol and may be stored at 4°C.

Paraffin processing

The automated processor takes the cassettes through a series of graded EtOH baths to dehydrate the tissues and then into xylene. Hot paraffin can then permeate the tissues:

  70% Methanol 1 hour (x1)
  90% Methanol 1 hour (x1)
  95% Methanol 1 hour (x1)
  100% Methanol 1 hour (x1)
  100% Ethanol 1 hour (x1)
  Xylene 1 hour (x2)
  Paraffin (65°C) 30 min. (x1)
  Paraffin (65°C) 30 min (x1) with vacuum (applied manually on our machine).

If the processor is to be run overnight it should be programmed to hold on the first EtOH bath and not finish until the next morning so the specimens do not sit in hot paraffin longer than the time indicated. This is important to keep the tissues from becoming hard and brittle. The vacuum helps to speed up the permeation of tissues by paraffin and helps get rid of any small air bubbles. Processed tissues can be stored in the cassettes at room temperature indefinitely.

Embedding tissues in paraffin blocks

Tissues processed into paraffin are melted by placing them in 65°C paraffin bath for 15 minutes. Block molds are warmed on a hot plate. Hot paraffin is poured into the mold and then the melted tissues are placed in the mold. Heated forceps are used to prevent the tissues from sticking. When the tissue is in the desired orientation the mold is placed on a cooling plate and the cassette backing is placed on top as a lid. After 30 minutes the hard paraffin block can be popped out of the mold. Tissue blocks can be stored at room temperature for years.

Sectioning tissues

Blocks to be sectioned are placed face down on an ice block for 10 minutes. Turn on the water bath and replace it with fresh deionized water (DEPC treated water must be used if in situ hybridization will be performed on the sections). Place a fresh blade on the microtome if necessary. Insert the block into the microtome chuck so the wax block faces the blade and is aligned in the vertical plane. Set the dial to cut 4 µM sections. "Face the block" by cutting it down to the desired tissue plane and discard the paraffin ribbon. If the block is ribboning well then cut another four sections and pick them up with forceps or brush and float them on the surface of the water bath. Float the sections onto the surface of clean glass slides. If the block is not ribboning well then place it back on the ice block to cool off and hydrate more.

Place the slides with paraffin sections in a 65°C oven for 20 minutes (so the wax just starts to melt) to bond the tissue to the glass. Slides can be stored overnight at room temperature.

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).

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: 0.3% (v/v) NH4OH (1 L H2O 3 mL 28% w/v NH4OH stock).
   

Procedure: Dip 20x in each solution unless otherwise indicated.

  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 (x2)
  100% EtOH (x2)
  Xylene 3' (x3)



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.