Staining, Nuclear and Cytoplasmic Lecture

This is tightly wound protein that is not actively being used or replicated

Heterochromatin

Heterochromatin is a stainable or nonstainable component?

Stainable

This is a loosely bound protein and is actively being used and replicated.

Euchromatin

Euchromatin is a non-stainable or stainable component?

non-stainable

Why can eosin stain the nucleolus?

Due to dense protein content

Hematoxylin stain acidic structures:

Blue

Eosin stains basic structures:

Pink

Covalent bonds:

share electrons STRONG

Ionic bonds:

positive is attracted to negative STRONG

Hydrogen bonds:

When covalently H+ attracted to strong electro-negative charge

WEAK

Van der Waal bonds:

Electrostatic charge

VERY WEAK

A physical stain is purely done by:

ABsorption

What is absorption?

Passing of dye molecule from the solution to the tissue component to be demonstrated

During the absorption, the stained tissue becomes what color compared to the dye solution?

THE SAME COLOR AS THE DYE SOLUTION

Chemical staining occurs by:

ADsorption

H and E staining is done by what kind of staining?

Adsorption

What occurs during adsorption?

A chemical union or reaction between a dye and stained substances

It occurs by ions within stain solutions being attracted to and influenced by ions in tissues

Dye molecules precipitate on certain cellular components

What is a metachromatic staining mechanism?

A change in color

a SINGLE dye reacts with tissue cells and stains them different colors

What is a polychromatic staining mechanism?

Contains 2 or more dyes

Dye solutions stain tissue components different colors by a phenomenon other than metachromasia

During absorption, molecules are:

drawn into material

During adsorption, molecules:

adhere to the surface

Eosin is acidic and has a:

negative charge

Eosin is what kind of dye?

Anionic

Eosin is attracted to what kind of structures?

Basic (+) cytoplasmic proteins

cytoplasmic granules

connective tissues

RBC

Nucleolus

Hematoxylin is a basic dye with what kind of charge?

Positive

Hematoxylin is a:

Cationic dye

Hematoxylin is attracted to:

Acidic (-) nucleus

heterochromatin

ER in cytoplasm

Acidophilic tissues are:

attracting acid dyes (anionic)

Basophilic tissues are:

attracting basic dyes (cationic)

Acid and basic dyes do not relate to pH, but to:

the charge (negative or positive)

What is the IEP (Isoelectric point)?

Point where positive and negative charges are equal

There is no migration between charged proteins at what pH?

6

Ideal cytoplasmic staining, the pH should be between:

4-5

If the pH is below 6, proteins have a ____ net charge and attract ___ dyes like eosin.

positive, anionic (-)

If the pH becomes too low, what occurs?

No longer charged

can bind non-specifically bind to tissue

If above pH, proteins have a ___ charge and will NOT attract ____ dyes (eosin)

negative, anionic

Eosin will not attach to a:

cytoplasmic protein

Erythrocyte staining by eosin is

Darkest

Collagen, muscle staining by eosin is

intermediate

Cytoplasm, staining by eosin is

lightest

What can be added to give more vibrant colors?

Phloxine

Hematoxylin is what kind of dye and where does it come from?

Nuclear dye from logwood tree

Hematoxylin must be___ to be used as a dye

Oxidized, turn into hematein

What is used as the link for hematein?

metal mordant

How does a hematoxylin stain work?

Use metal mordant to create dye lake (aluminum, iron, chromium)

dye lake stains tissue (optimal 2-2.5)

Provide a net positive charge for better binding to negative sites like the nucleus

Then blued to adjust pH 5 to change the solubility of the dye lake

Convert red complex of the nucleus to an insoluble blue color

EOSIN THAT TURNS THE BLUE NUCELI A MORE PURPLE COLOR

At the pH 2-2.5:

Favors selective nuclear stainig

At the pH 2.5-2.8:

There is a potential for light coloration of mucus (goblet)

A pH higher than 2.8 will cause:

Non-selective staining

What are the steps of staining?

Xylene

Graded alcohols

Water

Hematoxylin

Water

Clarifier

Water

Bluing

Water

Alcohol

Eosin

Alcohol

Xylene

Mounting media

When is a quality control slide ran?

at the beginning of each shift or when the stainer is changed

What can happen if the slides dry out before coverslipping?

Cornflaking

Why do solutions need to be covered when not in use?

Prevents atmospheric moisture from getting into absolute alcohol

prevent evaporation

Staining problems could stem from:

Poor fixation or processing

Water that contains minerals that could affect pH (iron, sulfur)

How thick are slides?

1-1.5 mm

What is the purpose of cover glass?

used for preservation of stained tissue sections

provide better microscopic magnification and examination

improve refractive index

What is coverslipping film?

used for automated coverslipper

use a cellulose triacetate film, coated with mounting media and is activated by xylene

NO DRYING TIME NECESSARY