Reticulin Fibers

How to Stain And Store Reticulin Fibers
Introduction
Under Light And Electron Microscopy
Procedure
Stains Used
Result

1. Reticular fibers By Selwyn Barreto

2. Introduction Reticulin fibers are fine delicate and frequently branch. They consist mainly of type collagen. They are found connected to the coarser and stronger collagenous fibers. Reticular fibers are abundant in smooth muscle. (eg uterus, intestinal muscle layer) Endoneurium and form the major supporting framework of the more cellular organs. (eg spleen, lymph node and red bone marrow) And constitute a network around the cells of parenchymal organs eg liver, endocrine glands. In these organs they are arranged in a 3D network and provide support to individual cells.

3. Light microscopy They Are not visible in section stained by routine hand e reticular fiber are weakly birefringent because of their lack of physical size and the masking effect of the interfibrillar substance they branch frequently.

4. Electron microscope The reticular fiber shows loosely packed fibrils surrounded by abundant carbohydrate rich interfibrillar material.

5. Staining reaction and demonstration of reticular fibers Reticular fibers may be demonstrated:- In paraffin section by any argyrophil type silver impregnation techniques because of their affinity for silver salts.  In frozen section by the periodic acid Schiff technique the carbohydrate matrix of the reticular fibers is responsible for both argyrophilia and pass positive staining property of reticular fibers and not due to the fibrillar elements of the fibers.

6. Stains for the reticular fibers Reticulin is procollagen and is finer whereas collagen fibers are coarse. Reticulin and collagen are basically similar reticulin fibers stain black with reticulin stain but is stained with collagen stain.

7. Techniques of demonstration By 2 techniques:- Dye techniques Metal techniques

8. Dye techniques Dye techniques using dye as a means of staining for demonstration of reticular fibers is not completely reliable and the density of stain being insufficient to resolve the fine fibers.

9. Metal techniques In most of the techniques there is ppt of silver from silver nitrate by sodium hydroxide( NaOH ), potassium hydroxide( KOH ), ammonium hydroxide( NH4OH ) or with lithium carbonate( Li2CO3 ), sodium carbonate ( Na2CO3 ). The composition of the silver solutions varies but all have in common the silver in alkaline solution.

10. Staining used for reticulin stain Gordon and sweet method for reticular fibers Gomoris silver impregnation method for reticular fibers H&E Van Gieson’s Stain  

11. Principle The aldehyde group of reticulum fibers reduce the colourless silver complex to a dark brown silver oxide which is precipitated on the reticulin fibers. The the silver oxide is reduced by a solution of sodium thiosulphate.

12. Fixative 10% neutral buffered formalin 37% formaldehyde solution =10 ml Sodium Chloride(Nacl)(AKA Table Salt) =0.8 g Potassium phosphate monobasic( KH 2 PO 4 ) =0.4 g Potassium phosphate dibasic ( K 2 HPO 4 ) = 0.65 g Distilled water = 90 ml

13. Quality control Liver tissue is used asa good control tissue Also Other Tissue Such As:- Spleen Lymph Node

14. Solution for Gomoris silver impregnation Silver Solution 10% Silver Nitrate (Impregnation) =10ml Silver Nitrate ( AgNO3 )=10.0g Distilled Water=100.0ml   10% Potassium Hydroxide =40ml Potassium Hydroxide( KOH ) =10.0g Distilled Water =100.0mL Strong Ammonia(NH 4 OH) =10 ml Distilled Water

15. 1% Potassium Permanganate (For Oxidation) Potassium Permanganate( KMnO₄ ) =5.0g Distilled Water =100.0mL 2% Potassium Metabisulfite (Decolouring Agent) Potassium Metabisulfite( K2S2O5 ) =10.0g Distilled Water =500.0mL

16. 2% Ferric Ammonium Sulfate (Sensitization)(AKA Iron Alum) Ferric Ammonium Sulfate( NH4Fe(SO4)2 ) =10.0g Distilled Water =500.0mL 10% Formalin( CH 2 O ) (Reduction)   0.2% Gold Chloride (Toning) 1% Stock Gold Chloride Solution( AuCl3 ) =10mL Distilled Water =40mL

17. 2% Sodium Thiosulfate (Remove Unreduced Silver ) Sodium Thiosulfate ( Na2S2O3 )=10.0g Distilled Water =500.0mL Nuclear Fast Red (Counter Stain) Nuclear Fast Red =0.5g Aluminium Sulfate( Al2(SO4)3 ) =25.0g Distilled Water =500.0mL Van Gieson (Counter Stain) Eosin (Counter Stain)