Hematoxylene and Eosin stain

                  

Hematoxylin and Eosin staining of Tissue Section

.Dr. Swapan Kumar Biswas. (M.D.)

1.     Put the slide on hot plate/ flaming for few seconds ( to soften paraffin )

2.     Then immediately dip slide in XYLENE for 5 min.(Minimum) to dissolve paraffin. After taking slide out, put some fresh xylene (Few drops) on the section to washout remaining paraffin.

3.     Wash slide with 100% alcohol in slanting way over both side of slide to remove xylene

4.     Then wash the slide with descending grade of alcohol ie, 90%,-70%- 50%-(2 min each), to hydrate the slide

5.     Ultimately put the slide in water for 5 min

6.     Bring up the slide from water and put in Koplins jar containing Harris’ Hematoxylene for 5 min. ( in modified method put hematoxylene on tissue section for 5 min)

7.     Keep slide in rectangular koplin jar in running water for 5 min.

8.     Dip 6the slide for 2/3 times in 1% acid alcohol (1% HCL),for differentiation (To remove excess stain in cytoplasm. colour  appear pink.

9.     Keep the slide in running water again till it become blue colour (Colour of the nucleus)

10.                        Put 1% aquous eosin over slide for 2 min.

11.                        Dip in water for 2 min, (in running water) or more if staining is more.  ( 30 SEC. IN CASE OF SPIRITE EOSIN SOLUTION)

12.                        Then dehydrate slide by putting ascending grade of alcohol ie, 50%-70%-90%-Absolute alcohol  for 2 min each.

13.                        Dry in air.

14.                        Dip the slide in clean xylene for 1 min.

15.                        Mount in Canada Balsam/DPX

16.                       

For practical purpose, blotting is done by tissue paper in place of  sl. 12

SEE IN MICROSCOPE.