H&E Staining Procedure PDF Free Download

H&E Staining Procedure PDF Download Info

Hematoxylin and Eosin Stain (H&E) Staining Procedure PDF Download Hematoxylin And Eosin (H&e) Is Still The Gold Standard For Most Diagnoses Where One Wants To See Minute Details Of Cellular And Tissue Structures. Variability In Intensity Is Generally Due To Pathologist Education And Preference.

H&E Staining Procedure PDF

In Reality, Nearly All Educational Material Uses H&e Images Because It Provides A Broad Spectrum Of Cytoplasmic, Nuclear, And Extracellular Matrix Features. This Stain Is Still In Use Today; Its Use Has Not Changed In Over A Hundred Years. It Is Simple And Essential.

Steps of H&E Staining

Hematoxylin and Eosin (H&E) staining is a widely used technique in histology to visualize the structure of tissues. Here are the detailed steps:

1. Fixation

• Fix tissue samples to preserve structure and prevent decay (commonly with formalin).

1. Dehydration

• Gradually dehydrate the tissue using increasing concentrations of alcohol (ethanol).

1. Clearing

• Clear the tissue with a solvent like xylene to make it transparent and prepare it for embedding.

1. Embedding

• Embed the tissue in paraffin wax to provide support for thin sectioning.

1. Sectioning

• Cut thin sections (about 4-5 micrometers thick) of the paraffin-embedded tissue using a microtome.

1. Mounting

• Mount the sections onto microscope slides.

1. Deparaffinization

• Remove paraffin by immersing slides in xylene.

1. Rehydration

• Rehydrate the tissue sections by passing them through a series of decreasing alcohol concentrations, ending in water.

1. Staining with Hematoxylin

• Stain the tissue with hematoxylin to bind to acidic structures, mainly nucleic acids, staining them blue or purple.

1. Rinsing
   • Rinse the slides in water to remove excess hematoxylin.
2. Differentiation
   • Differentiate in acid alcohol (if needed) to remove excess background staining, then rinse in water.
3. Bluing
   • Immerse slides in a bluing agent like ammonia water to enhance the hematoxylin staining.
4. Counterstaining with Eosin
   • Stain the tissue with eosin to bind to basic structures, mainly proteins, staining them pink.
5. Dehydration
   • Dehydrate the stained sections through increasing alcohol concentrations.
6. Clearing
   • Clear the sections with xylene.
7. Mounting
   • Mount the slides with a coverslip using a mounting medium.

Steps of a Basic Staining Procedure

A basic staining procedure typically involves the following steps:

1. Fixation

• Preserve the tissue or cells with a fixative.

1. Permeabilization

• Make the cell membrane permeable (if needed) to allow stain entry.

1. Staining

• Apply the stain to the tissue or cells.

1. Rinsing

• Rinse off excess stain with a buffer or water.

1. Mounting

• Mount the sample on a slide and cover with a coverslip.

Two Stains Used in an H&E Stain

1. Hematoxylin
2. Eosin

Principle of Eosin Stain

Eosin Is An Acidic Dye That Will Tend To Link To Basic, Or Alkaline, Structures Of The Cell And Stain Them. The Idea Behind Eosin Staining Is To Create A Reaction With The Positively Charged Elements Of The Cell To Generate A Pink Or Red Color Within The Extra-cellular Matrix, The Cytoplasmic Proteins, And Red Blood Cells.

This Acts In Counter-staining Contrast With Hematoxylin, Which Labels Acidic Structures, Mostly Nucleic Acids, A Blue Or Purple-colored Stain.