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H&E Stain Protocol: A Standard Procedure for Tissue Staining in Histopathology, Summaries of Medicine

University of DerbyMedicine

The hematoxylin & eosin (h&e) staining protocol used in histopathology for the routine staining of cationic and anionic tissue components. The protocol involves the use of specific reagents and solutions, and the staining procedure is detailed step by step. The principle of the protocol and the specimen required are also explained.

What you will learn

  • What type of tissue is required for the H&E staining protocol?
  • What is the purpose of the H&E staining protocol?
  • What are the steps involved in the H&E staining procedure?
  • What are the components of the H&E staining solutions and how are they used?
  • What is the purpose of the H&E staining protocol in histopathology?

Typology: Summaries

2021/2022

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WashingtonUniversitySchoolofMedicine
NeuromuscularLab
CAP:1923316
CLIA:26D0652044
NY:PFI3499
H&Eprotocol.docx
1997
HEMATOXYLIN & EOSIN (H & E) STAIN PROTOCOL
PRINCIPLE:
This protocol is applied in the routine staining of cationic and anionic tissue
components in tissue sections. This is the standard reference stain used in the study of
histochemical tissue pathology.
SPECIMEN REQUIRED:
Snap frozen human striated muscle. (Use the 2-methylbutane freezing method)
METHOD:
Fixation: None. Use snap frozen tissue.
Technique: Cut 10 - 16 micron (12 µm) sections in cryostat from
snap frozen biopsy. Attach first and last sections to a
Superfrost Plus microscope slide.
Equipment:
Ceramic staining rack - Thomas Scientific #8542-E40
Columbia staining dish - Thomas Scientific #8542-C12
Columbia staining dish(jar) - Thomas Scientific #8542-E30
Forceps Latex gloves
Reagents:
Reagent alcohol - HPLC Fisher A995-4 or histological A962,
FLAMMABLE store at room temp. in a flammable cabinet
Eosin Y, disodium salt (Sigma #E-6003, store at room temperature)
Harris Hematoxylin Stain, acidified (Lerner Laboratories #1931382)(R.T.)
Permount - Fisher SP15-100, FLAMMABLE; HEALTH HAZARD
Xylenes (Fisher #HC700-1GAL, FLAMMABLE
Solutions:
I. Eosin Y, 1 % aqueous (store at room temperature)
Eosin Y dye 1 g
Deionized water 100 ml
pf3

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Download H&E Stain Protocol: A Standard Procedure for Tissue Staining in Histopathology and more Summaries Medicine in PDF only on Docsity!

Neuromuscular Lab CAP: 1923316

CLIA: 26D NY: PFI 3499

H&E protocol.docx

HEMATOXYLIN & EOSIN (H & E) STAIN PROTOCOL

PRINCIPLE:

This protocol is applied in the routine staining of cationic and anionic tissue components in tissue sections. This is the standard reference stain used in the study of histochemical tissue pathology.

SPECIMEN REQUIRED:

Snap frozen human striated muscle. (Use the 2-methylbutane freezing method)

METHOD:

Fixation: None. Use snap frozen tissue.

Technique: Cut 10 - 16 micron (12 μm) sections in cryostat from snap frozen biopsy. Attach first and last sections to a Superfrost Plus microscope slide.

Equipment: Ceramic staining rack - Thomas Scientific #8542-E Columbia staining dish - Thomas Scientific #8542-C Columbia staining dish(jar) - Thomas Scientific #8542-E Forceps Latex gloves

Reagents:

Reagent alcohol - HPLC Fisher A995-4 or histological A962, FLAMMABLE store at room temp. in a flammable cabinet Eosin Y, disodium salt (Sigma #E-6003, store at room temperature) Harris Hematoxylin Stain, acidified (Lerner Laboratories #1931382)(R.T.) Permount - Fisher SP15-100, FLAMMABLE; HEALTH HAZARD Xylenes (Fisher #HC700-1GAL, FLAMMABLE

Solutions:

I. Eosin Y, 1 % aqueous (store at room temperature) Eosin Y dye 1 g Deionized water 100 ml

Neuromuscular Lab CAP: 1923316

CLIA: 26D NY: PFI 3499

H&E protocol.docx

  1. Harris Hematoxylin, acidified (store at room temperature) Filter (Baxter #F2217-150, Grade 363, Qualitative) before use
  2. Alcohol 50 % reagent alcohol ~50 ml deionized water ~50 ml
  3. Alcohol 70 % reagent alcohol ~70 ml deionized water ~30 ml
  4. Alcohol 80 % reagent alcohol ~80 ml deionized water ~20 ml
  5. Alcohol 95 % reagent alcohol ~95 ml deionized water ~ 5 ml

Staining Procedure:

  1. Place the slides with section in a metal staining rack.
  2. Immerse sections in the filtered Harris Hematoxylin for 10 seconds.
  3. Remove rack to a beaker with tap water.
  4. Exchange tap water until the water is clear.
  5. Immerse sections in EOSIN stain for ~30 seconds.
  6. Remove rack to a beaker with tap water.
  7. Exchange tap water until the water is clear.
  8. Dehydrate in ascending alcohol solutions (50%,70%,80%,95% x 2, 100% x 2) in Columbia staining dish(jar)s - Thomas Scientific #8542-E.
  9. Clear with xylene (3 - 4 x ) in Columbia staining dish(jar)s - Thomas Scientific #8542-E30.
  10. Mount coverslip onto the section on glass slide with Permount. or other suitable organic mounting medium.