View source for Histology:Chapter 1:Overview

== Overview of Tissue Preparation and Staining for Microscopy ==

1.  '''Obtaining tissues''' - Human material is obtained at autopsy or from surgical biopsies. 

2.  '''Fixation''' - To preserve the tissue, it is placed immediately in a fixative which acts to preserve the cell and tissue constituents in as lifelike a manner as possible after death.  In postmortem tissue, considerable autolysis may have occurred prior to fixation.  Formalin (10%) is the fixative most often used by pathologists.

3.  '''Dehydration''' - The fixed tissues must be dehydrated in order to embed them in paraffin for sectioning.  Water is removed from the tissues by passing them through a series of increasingly concentrated solutions of alcohol.

4.  '''Clearing''' - Absolute alcohol is not miscible with paraffin.  Thus, the alcohol must be removed from the tissue and replaced with an agent that mixes with molten paraffin. The most commonly used clearing agent is xylene.  The xylene makes the tissues translucent or “clears” them.

5.  '''Embedding''' - Following clearing, the tissue is placed in the embedding agent, molten paraffin, and allowed to steep until the tissue is thoroughly infiltrated by the embedding medium.  The preparation is then cooled, the paraffin solidifies, and the block of tissue can now be cut with a minimum of distortion.  The paraffin infiltrates the interstices of the tissue and thus provides internal support as well as external support for sectioning.

6.  '''Sectioning''' - The tissue is now cut into very thin slices, usually 5 to 7 m, with a microtome.  The sections are then mounted on glass slides and stained.

7.  '''Staining''' - For morphologic study, it is necessary to create color contrasts in the tissues by staining.  Certain terms are used to distinguish the staining reaction of a cell.  The term basophilic indicates that the structure can be stained with the basic dye hematoxylin. All nuclei are basophilic.  Cytoplasmic elements may be either basophilic, acidophilic or, neutral.   Eosin is the most commonly used acid stain and any acid components that stain positive with eosin are termed eosinophilic.

8.  '''Other stains''' used in preparing slides – Most slides for histology and pathology are stained with Hematoxylin and Eosin (H&E).  Additional staining techniques are utilized to demonstrate specific characteristics of tissues.  In any staining process variations in the tissue and the technical procedure may lead to minor color modifications in individual slides, but, in general, the reactions are as stated below.

a.	'''Masson’s Trichrome stain''' (hematoxylin, acid fuchsin, and aniline blue):  nuclei stain black or dark blue; cytoplasm stains red by the acid fuchsin; reticular and collagen fibers stain blue with aniline blue.
b.	Gomori’s Trichrome stain:  Another version of a trichrome stain that stains nuclei - red-purple; normal muscle myofibrils - green-blue with distinct A and I bands; intermyofibrillar muscle membranes – red; and interstitial collagen - green

c.	'''Periodic acid-Schiff’s reagent (PAS)'''.  The PAS method stains glycogen, mucin, connective tissue fibers, and other structures that contain carbohydrates, pink, red, or maroon.  The periodic acid converts adjacent 1, 2 glycol groups to aldehydes and the basic Fuchsin of Schiff’s reagent stains the aldehydes.  Sometimes Hematoxylin is used as a counter stain giving you a PASH.

c.	'''Silver stain'''.  This special procedure employs silver nitrate to specifically demonstrate reticular fibers, neurofibrils of neurons and granules in enteroendocrine cells.  These structures are stained black whereas other tissue components may take on a faint gray background stain without revealing detail.

d.	'''Toluidine blue'''.  Used to demonstrate granules in mast cells.  Nuclei are deep blue; mast cell granules are reddish-purple.

e.	'''Verhoeff-Van Gieson stain (VVG)'''.  This method is used for identifying elastic fibers in tissues such as skin, aorta, etc.  The elastic fibers will be stained blue-black and background will be stained yellow.