Histology Lab Techniques Lecture

 

As soon as tissue is removed from animal it begins to degenerate due to bacterial destruction and autolysis - to study tissue in its most natural state these processes have to be inhibited and the tissue preserved - a process called FIXATION.

 

1) Light Microscopy - usually aldehyde based fixative - formalin (formaldehyde) or in combination with other chemicals - i.e. Bouins - most commonly used general fixative

 

2) special techniques of Histochemistry (localization of enzyme activity) or Immunofluorescence (antigen detection with antibodies)

 

a) Aldehydes if possible (best structure) but they cross link proteins which can inhibit enzyme activity or change antigen structure = try others

 

b) Alcohol fixation - poor structure, but good enzyme activity and good antigen structure (no cross linking).  Alcohol is usually the fixative used to prepare blood smears

 

c) Freeze tissue (in liquid nitrogen) - poor structure, but good enzyme activity/antigen structure - go directly to sectioning on a cryostat

 

3) Electron microscopy - fixative made in physiological buffers

 

a) Glutaraldehyde - dialdehyde structure produces excellent fixation

 

b) Osmium tetroxide (OsO4) - EM "post-fixative" - follows glutaraldehyde and stabilizes lipids in cell, gives some electron density to tissue

 

Rules for good fixation:  (practical instructions)

 

1) use small pieces of tissue

 

2) use large volume of fix - 10x volume of tissue.

 

3) get tissue into fix FAST - keep cold (on ice) and wet (in buffer) during dissection - always wash off excess blood with buffer before fix

 

Therefore, when choosing a fixative you must consider what you need the specimen for - general structure (Bouins or formalin fixed), immunoflourescence (alcohol fixed or frozen tissue); TEM (aldehyde fix w/ Osmium post fix), etc.

 

 

Next, to section the fixed tissue thin enough to “see through” it, it must be made into a homogeneous block - process called EMBEDMENT


 

EMBEDDING TISSUES

 

For Light Microscopy:

 

1) Remove water from tissue = dehydration and clearing

 

            a) dehydrate with a graded series of alcohol (70 → 85 → 95 → 100%)

 

            b) ETOH and paraffin do not mix - Xylene is soluble in both and used to clear ETOH out of tissue

 

2) infiltrate w/ a homogeneous medium (paraffin) for sectioning - fills in all lumens so structure not crushed

 

a) place liquid paraffin into mold

 

b) drop in tissue and orient as desired - cross- vs longitudinal-section

 

            c) add label and allow to harden

 

 

For TEM, the process is basically the same:

 

1) After fixation, tissue is dehydrated with ETOH

2) The tissue is "cleared" with different organics soluble in ETOH and plastic

3) infiltration is done by incubation in a mix of organics and unpolymerized plastic resin

4) tissues are oriented in molds filled with plastic resin and harden in an oven for days.

 

SECTIONING

 

To produce thin sections, embedded tissues are sectioned on a microtome

 

a) 7 micron sections for LM - placed on glass slides

 

            b) 70 millimicrons - placed on metal grids for TEM

 

 

STAINING

 

Light Microscopy

 

1) Basic reaction of stains = attraction of opposites:

 

a) Tissues that stain with a basic stain = BASOPHILIC (stain acid component - ie. Nuclei or RER in secretory cells)

 

b) Tissues that stain with an acidic stain = ACIDOPHILIC (stain basic component - i.e. “Normal” cytoplasm)

 

To stain, sections must be rehydrated first (reverse clearing/dehydrate sequence back to water).  There are many different histological stains.  Some of the most common, and ones you in this class are the following:

 

 

1) Hematoxylin & Eosin (H & E) - most common stain - good for general structure

 

            a) nuclei = blue  (basophilic) → Hematoxylin

 

            b) cytoplasm = pink (acidophilic) → Eosin

 

2) Connective tissue stains - both employ a nuclear, cytoplasmic, and a third stain specific for matrix

 

            a) Masson's trichrome

 

            b) Mallory's triple C.T. stain

 

3) Silver Impregnation

 

a) specificity provided by what silver is complexed to and pH of staining solution

 

            b) used to trace nerves, stain golgi, reticular fibers

 

4) PAS (Periodic Acid Schiff's)

 

a) Schiff reagent a stain called Basic Fuchsin = specific stain for carbohydrates = PAS stain

 

5) Wright Stain for blood smears

 

a) uses azure blue stains to stain WBC granules basophilic or neutrophilic

 

b) used with eosin for RBS and eosinophilic

 

 

                             

For TEM - staining done with heavy metals = provide electron density to section - most commonly:

 

1) Uranyl Acetate

                                                                                      

2) Lead citrate