fixed Protocols

Category: Developmental Biology: Sea Urchin Developmental Biology

Preparation of fixed Sea Urchin embryos for immunocytochemistry and AP staining. Cebra-Thomas. Swathmore. - [Read Preparation of fixed Sea Urchin embryos for immunocytochemistry and AP staining]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

The method uses Trizol, a phenol-base reagent produced by Invitrogen. - [Read Preparation of RNA from Paraffin-Embedded Fixed Tissue Using Trizol Protocol]

Category: Immunohistochemistry Protocols: Blocking Protocols

Protocol for preparing a cell block from cell lines / cell suspensions. Includes: Live cell preparation; Fixed cell preparation. - [Read Preparing a Cell Block from Cell Lines Cell Suspensions Protocol]

Category: Drosophila Protocols: Drosophila Staining Protocols

Early embryos (0-17 hours or until cuticle formation) are treated with a mixture of organic solvents, formaldehyde, and alcohols, as described here. The cuticles of late-stage embryos are usually opened by sonication. Tissues from more advanced stages of development are normally dissected by hand and then fixed and stained in a standard paraformaldehyde/detergent combination - [Read Preparing Early Whole-Mount Drosophila Embryos for Immunostaining Protocol]

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

Frozen tissue sections show good preservation of tissue structure and antigens. The principle disadvantages of using them in immunostaining are that the specimens must be stored frozen, and a special microtome, known as a cryostat, is required. Also, many clinical specimens are not available in this form, and most classic histological descriptions of tissue structure and pathology are based on the use of paraffin-embedded sections of formalin-fixed material. - [Read Preparing Frozen Tissue Sections for Immunostaining Protocol]

Category: Drosophila Protocols: Drosophila Staining Protocols

Early and late embryos are treated with a mixture of organic solvents, formaldehyde, and alcohols. The cuticles of late-stage embryos (17-22 hours or until hatching) are usually opened by sonication, as described here. Tissues from later stages of development are normally dissected by hand and then fixed and stained in a standard paraformaldehyde/detergent combination. - [Read Preparing Late Whole-Mount Drosophila Embryos for Immunostaining Protocol]

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

Most histological studies are carried out on paraformaldehyde-fixed, paraffin-embedded tissue samples. Therefore, there is an extensive atlas of most tissues and organs prepared from these sources, and comparing the location of antigens to these data is immediately informative. The fixation and embedding procedures are harsh, however, and many antigens are not well preserved. - [Read Preparing Paraffin Tissue Sections for Immunostaining Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocols utilize T hybridomas to detect expression of peptide-MHC complexes, since these cells provide the most convenient, consistent, and flexible T cell readout systems for these purposes. If desired, antigen-specific T cell clones can be used in lieu of T hybridoma cells, but T cell clones often give poorer responses than T hybridomas to fixed APCs due to fixation-induced loss of costimulator function. - [Read Presenting Exogenous Antigen to T Cells Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

In preparation for FLIM-FRET analysis, the appropriate donor and acceptor components must be introduced into live or fixed cells. The method of introduction depends on the nature of the components and the state of the cells. For example, plasmid DNAs encoding a protein of interest fused to a variant of GFP may be introduced into live cells by transfection or microinjection, whereas labeled antibodies are delivered by microinjection. - [Read Probing Protein Interactions Using GFP and FRET Protocol]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Information on processing of microdissected tissue for molecular analysis. Includes: More than 10,000 Cells; Less than 10,000 Cells; Formalin-fixed; Paraffin-embedded Tissue; Other Fixatives; Timing. - [Read Processing of Microdissected Tissue for Molecular Analysis]

Category: PCR Protocols: Real-Time PCR Protocols

Protocol uses the Superscript II First-Strand Synthesis system for the generation of cDNA from total RNA.  RNA purified using TRIzol reagent (Invitrogen) or the methods described in Preparation of RNA Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifugation, Preparation of RNA from Paraffin-Embedded Fixed Tissue. - [Read Real-Time RT-PCR: cDNA Synthesis Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses the Superscript II First-Strand Synthesis system for the generation of cDNA from total RNA.  RNA purified using TRIzol reagent (Invitrogen) or the methods described in Preparation of RNA Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifugation, Preparation of RNA from Paraffin-Embedded Fixed Tissue. - [Read Real-Time RT-PCR: cDNA Synthesis Protocol]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

GUS is used as a tag to address nuclear localization whereas GFP is more versatile. GFP is detectable directly in living cells, GUS is only detected indirectly by staining of fixed tissue which may lead to artifacts or may obscure problems with protein solubility. In this protocol, protein localization is routinely assayed after particle-mediated transient transformation of onion epidermal cells. With this method it can be determined rapidly whether a given fusion protein is active and.... - [Read Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells]

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Protocol describes the transfer of RNA from agarose gels to neutral or positively charged nylon membranes, using upward capillary flow of neutral or alkaline buffers.  RNA becomes covalently fixed to positively charged nylon membranes during transfer in alkaline buffers.  However, treatment by UV irradiation or heating is required to fix RNA to neutral membranes. - [Read Transfer and Fixation of Denatured RNA to Membranes Protocol]

Category: Neuroscience Protocols: Histology Stains Protocols

Protocol stain for wet fixed cytology preparations: Papanicolaou Method. - [Read Wet Fixed Cytology Preparations Protocol]