Plant microtechnique and microscopy

Quick start. Paraffin embedding

Histology or in situ hybridization

Paraffin embedding

Microwave method

Glycol methacrylate embedding

Histology

Butyl/methyl methacrylate embedding

in situ hybridization or immunolocalization

Steedman's wax embedding

Immunolocalization

Chromosome squashes

Maize meiocytes

Hematoxylin staining

Microwave technique

Adjusting Kohler illumination

Microscopy. Magnifiers and microscopes

Compound microscopes

Principles of specimen illumination

Imparting contrast using inherent differences in index of refraction within the specimen

Confocal and wide-field deconvolution microscopy

Obtaining microscopic measurements

Chemical fixation. Types of chemical fixatives

The quality of fixation

Tissue storage

The mechanics of fixation

Coagulating fixatives

Noncoagulating fixatives

Recommendations for fixing plant issues

Tissue dehydration. General protocol

Dehydration using a graded solvent series

Ethanol, acetone

Rapid dehydration

Infiltrating and embedding tissues. Paraffin infiltrating and embedding

Polyethylene glycol and derivatives

Infiltration and embedding

Plastic infiltrating and embedding

Sectioning and mounting. General steps

Mounting blocks to microtome stubs

Factors influencing the quality of sectioning

The microtome knife

Setting up the microtome

Sectioning methods

Mounting sections to glass slides

Staining. Chemical classification of dyes

Dye chemistry

Microtechnique stains

General staining procedures

Staining equipment

Selected staining methods for plant microtechnique

Staining epoxy-embedded tissues

Reactive dyes

Cytogenetic techniques with squashed material

Cytogenetic stains for sectioned material

Staining pollen tubes

Mounting the coverslip

Alternate methods of microtomy. Free-hand sections

Vibratome

Freezing microtome (cryotome, cryostat)

Sliding microtome

Special methods. Tissue clearing

Macerating woody tissues

Macerating gymnosperm leaves for epidermal peels

Macerating nonwoody tissues

Sectioning carbonized wood samples

Microincineration

Bleaching tissues

Preserving color in whole mounted specimens

Repairing broken microscope slides

Microtechnique notes and problem solving. Tissue collection

Fixation

Fixatives for algae

Tannins

Hard materials

Sliding microtome

Clearing

Dehydration and infiltration

Embedding

Microtomy

Free hand sections

Staining

Histochemistry and cytochemistry. The relationship of the probe to the target

Cytochemical localization of cell components

Cytochemical localization of enzymes

Cytochemical fluorescence microscopy

Fluorescent dyes for specific applications

Intrinsic fluorescence

Background fluorescence and photobleaching

Localization of molecular targets in tissues. Immunolocalization

Example protocols for immunolocalization

Tissue printing to detect proteins and RNA in plant tissue

In situ hybridization

Autoradiography

Detecting incorporated radioactive elements in tissue sections

Whole-mount in situ hybridization

Tyramide signal amplification

TUNEL Assay for detecting DNA degradation and programmed cell death

Fluorescence in situ hybridization

Appendices. Toxics

Buffers

General information and common calculations

The mercury arc lamp in fluorescence microscopy

Manufacturers and vendors

Optics.