[Histonet] H&E LFB

[Patrick Dooley]

Here's my lab's SOP for an LHE. 7uM sections. Hope this helps!

1.    Deparaffinize and hydrate to absolute ethanol.

2.    Luxol Fast Blue 60° C oven 1hr.

3.    Cool to room temperature.

4.    Hydrate to water.

5.    2 - 3 dips reducer. The fresher the reducer the less number of dips
you need. You'll need to look under the microscope to determine an adequate
differentiation; the best is when you can still clearly see the axonal
processes extending into the neocortex but with not much staining in the
grey matter at all.

6.    Rinse in tap water until water is clear.

7.    Harris Hematoxlyn 10 min.

8.  Rinse in tap water until water is clear.

9.  Differentiate in acid alcohol.  This usually takes 1-2 dips. Again,
look under the microscope to detemine a sufficient stopping point to
differentiation.

10.  Rinse in tap water until water is clear.
11.  Eosin for 5 - 7 minutes.
12. Rinse in tap water until water is clear.
13. Dehydrate to absolute ethanol. This step adequately reduces the
background eosin in our lab, but you may need to leave/dip more in 95%
ethanol as that will differentiate it the best.
14. 10 dips in two separate changes of xylene.
15. Coverslip.

Solutions are as follows:

Luxol Fast Blue        10 g Luxol Fast Blue MBS

                                    1 liter 95% ethanol

                                    0.5 ml glacial acetic acid



Reducer                     10 g hydroquinone

                                    50 g Na2SO4

                                    1 liter distilled water



Acid Alcohol             10 ml conc. HOAc

                                    1 liter 70% ethanol



Eosin                          0.5% aqueous Eosin Y

                                    when first used, add 1 ml glacial HOAc
/ 400 ml eosin

Patrick Dooley, HTL(ASCP)

Research Tech I / Tissue Bank Coordinator

MGH Alzheimer’s Disease Resource Center

CNY 114 Rm. 2650

617-643-6994(office)