Staining 4.1: Basics Of Staining Flashcards
What is a stain and why do we do it?
- Staining imparts color and changes the refractive index of the tissue
- allows you to see details in tissue because tissue is transparent otherwise
Staining
The visual labeling of some biological entities by attaching or depositing it in its vicinity, a marker of characteristic color or form
Stain
The marker used
Affinity
Attractive forces that bind dye to tissue
- can get differential dye absorption based on affinity for tissue (multi shade staining)
Acidic and basic
Acidic : Anionic - negatively charged component
Basic: cationic - positively charged component
Natural Dyes
- Carmine
- Indigo
- Brazilian
- Orcein
- Saffron
- Hematoxylin
**CIBOSH**
Carmine
- Source: cochineal bug (break down shell)
- color : red
- Synthetic available : No
- have very little affinity for tissue, usually add metal salt eg iron, aluminum
- carmine + aluminum = mucicarmine
- stains mucin in goblet cells
Indigo
- Source: indigo plant
- color : blue
- Synthetic available : yes
Brazilin
- Source: Bark - Brazilwood
- color : red
- Synthetic available : yes
Red nuclear stain
Orcein
- Source: lichens (fungi)
- color : brown
- Synthetic available : yes
- boiled to create Orcinol
- orcinol + air + ammonia = orcein
- brown connective tissue stain (stains elastin)
- can get synthetic form now
Saffron
- Source: stamen of flowers
- color : yellow
- Synthetic available : No
- stains connective tissue yellow
Hematoxylin
- Source: logwood bark
- color : purple/blue
- Synthetic available : No
- biological name: hematoxylum campechianum
- hematoxylum = bloodwood
Hematoxylin + air = hematein
Hematein + Metal salt = Biological Stain
Synthetic dyes (all other dyes not part of CIBOSH)
Benzene Ring (colorless) reacts to form —————-> Quinone Ring (colored) base of all synthetic dyes
Chromophores
- Confer color on dye
- To alter shade add more of the same type of chromophore
To change color, and a different type of chromophore - different color
C=C, C=O, C=S, C=N, N=N, N=O, NO2
Auxochromes
- Imparts affinity of dye for tissue by salt beads/electrostatic forces
- May alter shade
- # of groups and charge of groups determines if dye is anionic or cationic
Cationic: NH3+
Anionic: COO- , OH-, SO3-, Phenol (-), halogens (-), metal salts(-)
what is a chromogen?
Chromogen = quinone + chromophores (no charge and doesn’t bind to tissue
Dye
Dye = quinone + chromophore(s) + Auxochrome (color and a charge)
Same dye, different auxochrome
eg. Eosin Y and Eosin B has different chromophores and auxochromes can have the same color although they have a different make up
Basic/ Acidic Dyes
Tissue charge: positive
Convention: basic
Dye it attracts: negative - acidic
Tissue classification: acidophilic (refers to TISSUE components, eg cytoplasm is basophilic)
Tissue charge: negative
Convention: acidic
Dye it attracts: basic
Tissue classification: basophilic
Leuko Dyes
Dyes that undergo oxidation/reduction to become colorless and then reduction/oxidation to have color return
E.g reduced magenta hydrocarbon + Shiffs reagent (sulfuric acid) = oxidized clear hydrocarbon
Mordants
Dye + Mordant = lake
- most auxochromes are ANIONIC
- to stain ACIDIC ANIONIC tissue components a chemical group with a POSITIVE charge must be added = MORDANT
Traditionally +2 or +3 metals are used
3 options:
- mordant applied to the tissue before the dye
- mordant applied to the tissue after the dye
- mordant and dye are applied to the tissue at the same time
See slides 25-27 (Staining 4.1) for illustration
How does dye bind to the tissue?
- Substantive - direct
- dye binds directly to tissue - Adjective - indirect
- tissue is treated first then dye is attached (eg thru oxidation/reduction first) - Impregnation
- metallic dye deposited onto the structure it is staining, not in - absorption
- dye is more soluble in the tissue than the solvent in which it is dissolved into (mostly done in lipid staining)- remember, my Abs don’t show because of my Lipids
Direct/indirect staining vs impregnation
Direct/indirect = same size, color changes due to tissue interactions with dye
E.g quick diff stain to detect H. Pylori (small and needs to be zoomed in on under microscope)
Impregnation = tissue component becomes larger due to addition of dye eg. Silver nitrate component layer
E.g Steiner to detect H. Pylori (makes it appear lager and easier to see under microscope)
Tissue: Stain interactions
Dye tissue/reagent tissue affinities
- Dye-tissue — mucicarmine (+ve) staining sulfates and carboxylated (-ve) epithelial acid mucopolysaccharides
-
Reagent-tissue — potassium ferrocyanide (CN-, -ve) being attracted to ferric ions (+ve) = Prussian blue
-Specific chemical reactions including :
A. Hydrogen bonding
B. Columbia interactions
C. Van der waals interactions
D Covalent bonding - Solvent- Solvent interactions
- tendency of hydrophobic groupings to come together in aqueous solutions, the key to fat staining
- dye has greater affinity for the lipids than isopropanol (dye jumps out of solution and into the tissue)
- Dye-Dye interactions: dye molecules can interact with each other
- can occur in solution or in tissue ‘
- metachromatic staining - the dye: tissue interactions result in one dye staining the tissue in different colors
E.g. toluidine blue - blue nuclei and purple mast cell granules
Other staining types
- Negative staining
- dye is taken up by everything but the target tissue; creates an outline of where the target is
I.e. Congo red staining for capsule - Vital/supravital staining
- usually fluorescent probes
- stains are injected into living animals to visualize biochemical or physiological properties of an organ or cell population
I.e lymphocyte tracking
Progressive staining
- place slide in dye , when properly stained, remove from dye
- tissue differentially picks up dye according to its affinity for different components
- usually a weak solution send - slower and more control
- prevents over staining
vs Regressive staining
- place slide in dye, over stain tissue and differentiate out background stain
- if you differentiate too long too much dye will be removed
- can use a stronger stain solution - only need control during differentiation
Methods of differentiation
- Opposite charges
- by going to the opposite side of Isoelectronic point (IEP) of components :
a. Cationic dyes are removed in weak Anionic solutions
b. Anionic dyes are removed in weak cationic solutions
E.g H&E staining : hematoxylin is diluted HCl (H+); eosin is ammonia water - Oxidation/reduction
- by reducting/ oxidizing the dye the interactions are between tissue components decrease
- e.g. Weil - excess mordant (+ve charge)
- the abundance of mordant in the solution will compete with the dye that is bound to the tissue and mordant
- mordant can bind directly to tissue or surround the dye to remove it so it is not bound to tissue anymore
How do dyes stay in the tissue?
- Dye has a higher affinity for tissue elements than for the subsequent solutions for staining or dehydration, clearing and coverslipping
- some dyes are pulled into subsequent steps eg crystal violent, methylene blue, light green
- basic dyes will stain acidic tissue components and vice versa
factors affecting staining
- Rate of:
a. Reagent uptake
b. Reaction
c. Reagent loss - Reagent used
- Number and affinity of binding sites
Example - multiple dyes in one staining solution = competition
A. - vely charged acidic dyes will have a high affinity for +vely charged structures and low affinity for - vely charged tissue structures
B +vely charged basic dyes will have a high affinity for -vely charge structures and low affinity for +vely charged tissue structures
Maximizing selective affinity thru altering pH of the staining solution
- the IEP of a protein is the point at which it is IEP neutral (can be polar but carries no net charge)
a. Decreasing pH of the staining solution will make the solution more acidic giving the proteins a net +ve charge
b. Increasing the pH of the staining solution will make the solution more basic, giving the proteins a net -ve charge
Isoelectronic point
description of a solution relative to a protein’s IEP
- if the IEP of a protein is X:
A. A basic solution has a pH>X
B. A acidic solution has a pH<X - acid or base can be any number not by the convention of acidic is less than pH 7 and basic is > pH 7
- in this instance acid/base are being utilized as a descriptive term to relate solution to the proteins IEP not to describe where a solution falls on the pH scale
IEP examples
Protein X has an IEP of 3.0
- staining solution pH 5.0 is more basic than protein X’s IEP
- staining solution pH 2.0 is more acidic in elation i protein X’s IEP
Zwitterion
Protein has a net charge of 0 at the IEP
Zwitterion examples
A. IEP pH = 4.0
Staining solution pH of 5.5
Is the solution acidic or basic
- solution is basic in relation to IEP; which means, protein will have an OH ion; takes away H from NH3+ to make H20;
- leaves NH2 on the ion, leaving the overall charge -ve
- protein is acidic (basophilic ; attracted to +ve dyes)
B. IEP pH= 9.0
Staining solution pH of 8.0
staining solution is acidic in relation to the IEP of protein
Meaning H+ are present to bind to the COO-; leaving overall +ve charge on the protein
- protein is basic (acidophilic, and attracted to -ve dye)
Other factors that influence staining
- Salts added to staining solution
- influences dye-tissue interactions - Dye concentration
- stronger solution = faster staining - Fixation
- fixed tissue stains better
- fixation alters overall protein charge and affinity for dye
Eg. Formalin (-vely charged) binds to +ve tissue components leaving a net -ve charge; basophilic tissue that wants a +ve dye
since formalin +vely charged and binds to negative components in tissue leaving a net + charge; tissue becomes acidophilic; needs a -vely charged basic dye
- Temperature of staining solution
- increased temp:
- increased brownian motion
- increased protein swelling and r-group exposure
- increased thermal energy - Time spent in staining solution
- more time in stain, more tissue staining - pH
- change ph, change rate and ability of dye to stain tissue - Blocking/extraction
- block or extract component you wont be able to stain it - Prove staining - Whole cells stain differently than sectioned cells
- Size of the cell structures vs. microns cut at
- changes the probability that the cell has been cut open
- to stain an intact cell, the dye must penetrate the plasma membrane
- cut open cells to give the dye more access to protein components
color index
- Color index numbers (C.I)
- unique identifiers for dyes based on their chemical structure
- event though a chemical may have 2 different names, the CI number will show if they are Sam/different structure
E.g. parasaniline and basic fuschin both refer to CI42500
Make sure to check before ordering dyes
