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

1. RETICULARFIBERS
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. STAININGREACTIONANDDEMONSTRATIONOF
RETICULARFIBERS
• 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. STAINSFORTHE
RETICULARFIBERS
• 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. TECHNIQUESOF DEMONSTRATION
By 2 techniques:-
• Dye techniques
• Metal techniques

8. DYETECHNIQUES
• 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. METALTECHNIQUES
• 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. STAININGUSEDFOR
RETICULINSTAIN
• 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(KH2PO4) =0.4 g
Potassium phosphate dibasic (K2HPO4) = 0.65 g
Distilled water = 90 ml

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

14. SOLUTIONFOR GOMORISSILVERIMPREGNATION
• 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(NH4OH) =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(CH2O) (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)