Eosin Y in Staining

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Questions and Answers

What is the primary reason for removing water from the stained section?

Because water is not miscible with the mounting agent (correct)

To allow the mounting agent to bond with the tissue

To preserve the stained tissue

To increase the refractive index for viewing under the microscope

What is the purpose of immersing the slide in a series of alcohol concentrations?

To prevent distortion of the tissue (correct)

To preserve the stained tissue

To remove impurities from the tissue

To increase the refractive index

Why is it important to observe the sections under the microscope after dehydration?

To check for impurities in the tissue

To determine if additional differentiation is needed (correct)

To assess the level of dehydration

To check for mounting agent residue

What is the role of xylene in the clearing step?

To remove alcohol from the section Signup and view all the answers

What is the purpose of raising the refractive index of the section?

To allow for better visualization of tissue details Signup and view all the answers

What is the composition of the mounting agent DPX?

Dibutyl phthalate, Polystyrene, Xylene Signup and view all the answers

What should you do if you observe incomplete clearing or dehydration after mounting in DPX?

Return the slides to the clearing agent and re-mount Signup and view all the answers

What is the ultimate goal of the procedures after staining?

To preserve the stained tissue permanently Signup and view all the answers

Why is Eosin Y the most widely used?

Because it is soluble in alcohol Signup and view all the answers

What is the purpose of adding acetic acid to the eosin solution?

To sharpen the staining Signup and view all the answers

What is the first step in the H&E staining procedure?

Dewaxing Signup and view all the answers

Why is dewaxing necessary in the H&E staining procedure?

To remove wax from the tissue section Signup and view all the answers

What is the purpose of hydration in the H&E staining procedure?

To prepare the tissue section for staining Signup and view all the answers

What is the consequence of excessive heat during dewaxing?

It damages the tissue Signup and view all the answers

What is the role of alcohol in the H&E staining procedure?

It removes the clearing agent from the tissue section Signup and view all the answers

What is the purpose of adding thymol to the eosin solution?

To inhibit the growth of fungi Signup and view all the answers

Study Notes

Eosin Y

Eosin Y is the most widely used due to its solubility in alcohol.
It is typically used as a 0.5 or 1% solution in DI water with a crystal of thymol added to inhibit fungal growth.
Adding 0.5 ml of acetic acid to 1000 ml of stain solution can sharpen the staining.

Staining Procedure for H&E

The procedure consists of 9 steps: dewaxing, hydration, hematoxylin, differentiation, bluing, eosin, dehydration, clearing, and cover-slipping.

Preparation of Wax Sections for Staining

Dewaxing is necessary to remove wax from sections, as wax hinders the reaction of dyes with tissues and renders them impermeable to stains.
Xylene is used as a clearing agent, and heat can be incorporated to speed up the reaction.
Sections must be dry before immersing them in xylene.

Hydration

Immerse sections in decreasing concentrations of alcohol (100% → 90% → 70% → water) to remove the clearing agent.
Replaced cloudy alcohol solutions with fresh ones.

Procedures After Staining

It is essential to mount the stained section with a mounting agent to preserve the tissue and increase the refractive index for viewing under a microscope.

Mounting Procedures

Dehydration: remove water from the stained section by immersing it in a series of alcohols (70% → 90% → absolute alcohol) to prevent distortion of the tissue.
Clearing: remove alcohol with xylene, which raises the refractive index of the section.
Mounting: use a mounting agent (DPX) to permanently preserve the stained section, ensuring no bubbles are trapped.