Staining 4.1: Basics Of Staining Flashcards

Question

Other staining types

Answer 1

1. 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 2. 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

Answer 2

- 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

Answer 3

- 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

Answer 4

1. 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 2. Oxidation/reduction - by reducting/ oxidizing the dye the interactions are between tissue components decrease - e.g. Weil 3. 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

Answer 5

- 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

Answer 6

1. Rate of: a. Reagent uptake b. Reaction c. Reagent loss 2. Reagent used 3. 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

Answer 7

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 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

Answer 8

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

Answer 9

Protein has a net charge of 0 at the IEP

Answer 10

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)

Answer 11

1. Salts added to staining solution - influences dye-tissue interactions 2. Dye concentration - stronger solution = faster staining 3. 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 4. Temperature of staining solution - increased temp: - increased brownian motion - increased protein swelling and r-group exposure - increased thermal energy 5. Time spent in staining solution - more time in stain, more tissue staining 6. pH - change ph, change rate and ability of dye to stain tissue 7. Blocking/extraction - block or extract component you wont be able to stain it - Prove staining 8. Whole cells stain differently than sectioned cells 9. 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

Answer 12

- 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