Reticulin

                Reticulin

Principle

1)   Preoxidation  with potassium permanganate followed by bleaching in oxalic acid inhibits the argyrophilia of nerve fibers.

2)   Sensitization  with Fe alum increases the affinity of reticulin fibers for

1.    Silver

3)   Reduction with formaldehyde solutions results in the reduction of silver  oxide to metallic silver and is deposited on the reticulin fibers.

4)   Toning with gold chloride increase the contrast of stained section .

5)   Fixing in sodium thiosulphate removes unreduced silver

6)   Treatment with potassium metabisulphate removes the excess of gold chloride

Preparation of  Ammoniacal Silver solution

Glassware's should be thoroughly cleaned and rinsed well with distilled Water. Ammonia should be pure and metal forceps should not come into contact with annoniacal silver solution.

1)   Take 1ml of 10% silver nitrate in a clean test tube .

2)   Add 2 – drops of 10% sodium hydroxide .

3)   A precipitate of silver hydroxide will formed .

4)   Add 28% ammonia drop by drop, until the solution is clear ( Silver oxide is formed ) .

5)   Add 10% silver nitrate drop by drop until you get a muddy colour

6)   Dilute with equal of distilled water.

7)      The solution should be prepared before starting the procedure,

Preparation of Potassium permanganate – Pink Color

          Potassium Permanganate    –       0.5 gm

           Distilled water                        -        100 ml

Preparation of 1% oxalic acid  - Color less

 

                     Oxalic acid                    -        1gm

                   Distilled water        -        100ml

         

Prepration of 10% Formalin

    40% Formaldehyde           -           10ml

     Distilled Water                 -           90 ml

Preparation of 0.1% Gold Chloride ( Straw yellow color)

           Gold Chloride                         -           0.1gm

           Distilled water            -           100ml

Prepration of  5% Sodium Thiosulphate ( Colourless)

           Sodium Thiosulphate            -           5 gm

           Distilled water                        -           100ml

Prepration of  5% Potassium Metabisulphate ( Colorless )

            Potassium Metabisulphate             -           5gm

            Distilled Water                                  -           100ml

Procedure

1.      Deparaffinise the section in xylene for 5-10 mnts

2.      Brin the section to water through descending series of alcohol.

3.      Oxidise in potassium permanganate for 2-3 mnts

4.      Wash in tap water

5.      Bleaching in oxalic acid for 2-mnts

6.      Wash in tap water

7.      Sensitize with iron alum for 5-mnts .

8.      Rinse well with distilled water

9.      Impregnate with ammonical silver solution for 2-mnts .

10. Wash well in several changes of distilled water .

11. Reduce in 10% Formalin for 1 mnts

12. Wash with tap water . Section should turn brown in color .After reduction with formalin

13. Tone with gold chloride for 10mnts . Section should turn to grey color .

14. Fix in sodium thiosulphate for 5-minutes

15. Wash with tap water .

16. Treat with potassium metabisulphate for 5-mnts

17. Wash with tap water .

18. Dehydrate , Clear and Mount .

Result ..

Reticulin fibre                        -           Black

Collagen                                 -           Purple Black