Tissue preparation for making histological slides: easy & short discussion

Tissue preparation

Under the light microscope, tissues are examined via a light beam that is transmitted through the tissue. But the tissue and organs are usually too thick for light to pass through them. For this region tissues have to sectioned to obtain thin, translucent sections. Before making the histological slide tissue has to pass though some preparation processes. Following are the steps of tissue preparations of histological slide.

Steps of tissue preparation for study 

1. Selecting parts of tissue: part of an organ selected and cut. The best thickness are 3-5mm

2. Fixation: The process of avoiding tissue damage from bacteria or autolysis (cell digestion by cells own enzymes) by the help of some chemicals (fixatives) is called fixative.

Name of fixatives: Formaline: Formaldehyde  is a gaseous substance but 40% of it soluble in water. In tissue prepation we uses  :10% formaldehyde(CH20) solution in water.

• Uses of formalin: used as a disinfectant or to preserve biological specimens.

•  Health hazards: short time exposure:  irritation of eye,skin, mucosa of nose and throat, it is  carcinogenic substance  but not teratogenic.
• Precurtion during use of formalin :
•  Exposure to mild formalin for 8 hrs /per day or 40 hrs in a week is safe. Formalin is not absorbed through skin so mild formalin can be handling for short time. After handling wash hand with soap and plenty of water for few min. For long term handling use gloves. Contamination of eye with formalin: wash eye with water for 15 min and consult with ophthalmologist.

Dehydration : after fixation, tissue must be dehydrate by graded series of ethanol and water, usually from the 70% to 100% 

Clearing : after dehydration, tissue must be clean with xylene.

3. Infiltration: after clearing  tissue must be infiltrate with paraffin.

 What is paraffin: for infiltration, tissue must place into a container containing liquid paraffin and place it  in an incubator at the temperature of 58-60 C 

4. After that tissue must be place in a cash for embedding or block formation
5. then sectioning with microtome : now block of the tissue cut by microtome ( a cutting instrument ) 

Paraffin block :Tissue within it  

Microtome machine

Thin section of tissue

 Removing extra paraffin from tissue section & picking up tissue on glass slide for staining 

4. Staining: unfortunately tissues are colourless can not be observe in microscope. So need to be stain. For staining rehydration is necessary. Rehydration is done by graded ethanol and water from 100% to 70%.
5. In routine staining hematoxilin and eosin

Name of the staining and its components:

Basophilic : Tissue components that stain more with basic dye is called basophilic

Acidophilia : tissue components that stain more with acidic dye is called acidophilic

Colour of different components of tissue by hematoxilic and eosin staining :
Hematoxylin is a dark blue or violet stain that is basic/positive. It binds to basophilic substances (such DNA/RNA - which are acidic and negatively charged).
DNA/RNA in the nucleus, and RNA in ribosomes in the rough endoplasmic reticulum are both acidic because the nucleic acid building blocks that come off the phosphate backbone are negatively charged. These form salts with basic dyes containing positive charges. Therefore, dyes like hematoxylin will bind to them and stain them violet.
Eosin is a red or pink stain that is Acidic / Negative. It binds to acidophilic substances (such as proteins - which are basic and positively charged).

6. Dehydration: after staining tissue need to be dehydrate again for long preservation.

7. Clearing: after dehydration tissue need to be clean with xylene. 
8. Amounting: DPX. This chemical place above the tissue and covered by coverslip for long term use.

Now a histological slide is ready for study.