histopathology in comparative glycan analyses: use of...

Histopathology in Comparative Glycan Analyses:

Use of Glycan Binding Probes in Histochemistry

Professor Nissi Varki

2019 July Glyco Boot Camp—Histochemistry

Histopathology in Comparative Glycan Analyses 1

Chapter 48 Essentials of Glycobiology 3rd Edition

Glycan-Recognizing Probes as Tools

Richard D. Cummings, Alan G. Darvill, Marilynn E. Etzler, and Michael G. Hahn.Published online: 2017.

Antibodies, lectins, microbial adhesins, viral agglutinins, and other proteins with carbohydrate-binding modules, collectively termed glycan-recognizing probes (GRPs), are widely used in glycan analysis because their specificities enable them to discriminate among a diverse variety of glycan structures.

The native multivalency of many of these molecules promotes high-affinity avidity binding to the glycans and cell surfaces containing those glycans.

This chapter describes the variety of commonly used GRPs, the types of analyses to which they may be applied, and cautionary principles that affect their optimal use.


BACKGROUNDThe first evidence that glycans were antigenic arose from the discovery of the human blood group ABOantigens (Chapter 14).

A key tool in these studies were plant lectins that by the mid-1940s had found widespread use in typing blood, because some were relatively specific for blood types and they could be easily purified and are stable (Chapter 31).

The discovery of the blood groups and the antibodies and lectins binding them indicated that such proteins could also be generally useful in identifying specific glycan sequences.Hundreds of different plant and animal lectins and other proteins with carbohydrate-binding molecules (CBMs) have now been characterized.

Thus, although monoclonal antibodies (mAbs) are often more specific for glycan determinants and bind with higher affinity, many plant and animal lectins and CBMs also have useful specificities for determinants beyond monosaccharides, have cloned sequences, are usually less expensive and commercially available, and have well-characterized binding specificities.

GBPs are also found in many other organisms (Chapters 31–36), including viruses and bacteria (Chapter 37), and reagents from these organisms are also being used in the field. The availability of GBPs and mAbs has helped to catapult the field of glycobiology into the modern era.


https://www.ncbi.nlm.nih.gov/books/n/glyco3/bm1/def-item/abo/

https://www.ncbi.nlm.nih.gov/books/n/glyco3/ch14/





LECTINS MOST COMMONLY USED IN GLYCAN ANALYSISMany of the lectins currently used as tools in glycobiology originate from plants, but some also come from animals (e.g., snails) or mushrooms.

Most were characterized initially by hapten inhibition assays, in which monosaccharides, their derivatives, or small oligosaccharides block binding to cells or other glycan-coated targets.

Such small-sized molecules that compete with binding of a lectin or antibody to a larger ligand are termed haptens.

Lectins are often grouped by specificity depending on the monosaccharide(s) that can inhibit their binding at millimolar concentrations and their distinct preference for α- or β-anomers of the sugar.

However, lectins within a particular specificity group may also differ in their affinities for different glycans.

The binding affinity (Kd) of lectins for complex glycans is often in the range of 1–10 µm, but for monosaccharides the affinity may be in the mm range.

For complex glycoconjugates with multiple determinants or multivalency, the binding avidity of lectins may approach nanomolar values.


Examples of Types of Glycan Determinants Bound with High Affinity by Different Plant Lectins

©2017 The Consortium of Glycobiology Editors, La Jolla, California

Chapter 48, Figure 4. Essentials of Glycobiology, Third Edition

Symbol Nomenclature for Glycans (SNFG)Buy the Book


Sialic acid binding lectins


Examples of Different Mammalian Glycan Antigens Recognized by Specific Monoclonal Antibodies




Histopathology in Comparative Glycan Analyses7

Examples of Different Uses of Plant and Animal Lectins, Carbohydrate-Binding Molecules, and Antibodies in Glycobiology




Histopathology in Comparative Glycan Analyses8

Useful links for methods in histochemistry and immunohistochemistry can be found on:

http://mousepheno.ucsd.edu/

For immunohistochemistry background and other info:https://www.sciencemag.org/custom-publishing/webinars/improving-tissue-sample-profiling-optimization-and-application

For lectin assays on frozen sections:https://www.jove.com/video/3928/using-unfixed-frozen-tissues-to-study-natural-mucin-distribution

Histopathology in Comparative Glycan Analyses


https://www.sciencemag.org/custom-publishing/webinars/improving-tissue-sample-profiling-optimization-and-application

https://www.jove.com/video/3928/using-unfixed-frozen-tissues-to-study-natural-mucin-distribution


Paraffin sections are placed onto slides and allowed to air-dry at room temperature


Steps for Lectin histochemistry using a humid chamber

1. De-Paraffinize sections in the Fume Hood (Xylenes are toxic inhalants)

2. Rehydrate in graded alcohol washes into washing buffer

3. Remove Endogenous Biotin (this is important when using using a biotin label since all

tissues have endogenous biotin, especially heart, kidney, liver)

4. Wash slides in slide holder, three times in washing buffer between each step

5. Make diluting buffer containing 10 mM Ca++ and 10 mM Mn ++(this is important for

certain lectins)

6. Overlay primary reagent at 1- 5 ug/ml (Diluted in diluting buffer-- in 200 ul per slide)

7. Incubate at room temperature for 30 minutes or overnight at 4 degrees in a humid

chamber, covered with parafilm to ensure contact with reagent to sections evenly, and to

ensure that they do not dry out)


De-paraffinize sections on slides in the fume hood

TBST

TBST

TBST

TBST


Washing Steps for Lectin histochemistry


Steps for Lectin histochemistry, biotin block


Steps for Lectin histochemistry: overlay with parafilm before incubation



Secondary steps after biotinylated lectin application:

1. Wash slides and overlay with secondary reagent and incubate in humid chamber for 30

minutes at room temperature in covered humid chamber

2. Overlay with Alkaline Phosphatase-conjugated Streptavidin and incubate in humid chamber

for 30 minutes at room temperature in covered humid chamber

3. Wash slides again and make Alkaline phosphatase Substrate buffer and test it

4. Overlay onto slides and cover and incubate for 5-10 minutes

5. Stop reaction if the negative control starts showing color

6. Wash slides and counterstain nuclei and coverslip for viewing and digital photomicrography


Tissue section: Frozen or deParaffinized

Tertiary reagent is used usually labeled with :

fluoresceinated compounds

or with an enzyme

Remove endogenous binding sites in tissue to prevent nonspecific binding

Wash off unbound tertiary before adding substrate or before mounting to view or before further amplification

CY2 , FITC

AMCA

PE, CY3

HRP Alk.Phos

DAB, AEC, red , SG, VIP Blue, Red (also fluoresces)

Primary

Secondary

Tertiary


Effect of different fixatives on preserving epitopes


Histological Differences between Human and Mouse organs

Differences between Frozen and Paraffin samples


Un-inflated mouse lungs: Cannot be examined adequately by microscopy

Caveats in histology processing of mouse organs

Mouse brains have to be perfusion fixed

Lung lobes are to be separated and placed into labeled cassettes

Mouse Spleens and Skin and Pancreas have to placed between sponges while fixing

Mouse Livers have to be sliced thin and have to be placed in cassettes for fixing

Do not fix for longer than 24 hours if immunohistochemistry is planned

HISTOPATHOLOGY in Comparative Analyses

Outline of Specific Aims:The overall goal of the Histopathology Core in Comparative Analyses is to

enhance and facilitate the research of the individual Research Units by providing

the following histological services :

§ Training and assistance with harvesting organs and processing of tissues

§ Tissue sectioning

§ Standard staining, special stains, lectin histochemistry and immunohistology.

§ Training and assistance with microscopic analysis and interpretation of

histological studies


Detail of Services offered by the Histopathology Core to assist with Comparative Analyses:

Training and assistance with harvesting organs and processing of tissues:- Harvesting of mouse tissues, using optimal methods.- Freezing of organs, tissues and cells.- Fixing, orienting, processing and embedding in paraffin, of organs, tissues and cells.Tissue sectioning:- Preparation of paraffin sections. - Preparation of frozen sections and training as needed.Standard staining, special stains, lectin histochemistry and immunohistology.- Standard and specialized histochemical staining and training as needed.- Lectin histochemistry with controls and inhibitors.- Immunohistological staining, of frozen/paraffin sections with enzyme/fluorochrome-labels.- Antigen retrieval and signal amplification methods.- Assays for apoptosis or for proliferating cells. Training and assistance with microscopic analysis and interpretation of histological studies- Advice on experimental design of histological studies.- Assistance with microscopic analysis, histological evaluation, interpretation, and photo-documentation.



Histopathology analyses on Array of mouse tissues on slides

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