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Upstate - Procedures and Protocols - Tips for Immunohistochemistry with Fixed Paraffin-Embedded Tissues
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Upstate Cell Signaling Symposium
The first annual Upstate Cell Signalling Symposium will take place in Dundee, Scotland (UK) from 6 - 9th June 2004 and is entitled:

Regulation and Therapeutic Potential of the PI 3-kinase/PKB Signalling Pathway.

The symposium is co-sponsored by Sir Philip Cohen and Prof. Dario Alessi (The MRC Protein Phosphorylation Unit) in conjunction with Upstate.

Participation is strictly limited to 300 delegates, so early registration is recommended.

Tips for Immunohistochemistry with Fixed Paraffin-Embedded Tissues

  • To allow interpretation of the staining patterns, isotype and system controls should always be run and must be matched to the isotype of each primary antibody to be tested.
  • For the best tissue adherence, use positively charged slides (Poly-L-lysine or silane coated slides are also acceptable) and use deionized water in the waterbath when cutting paraffin sections.
  • Antigens are affected differently by the various methods of pretreatment. Our products are quality control tested by immunohistochemistry on paraffin-embedded sections. It should be noted that the length of pretreatment will vary for different antigens and different tissues and should be optimized by the investigator.
  • Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used in diluting the peroxidase substrate.
  • If an alternative substrate to diaminobenzidine (DAB) is used, the optimal development conditions (time, concentration and pH) should be established by the investigator. [Note: Some counterstains may not be compatible with some of the peroxidase substrates and some substrates other than DAB may require aqueous mounting media.]
  • During the staining procedure, do not allow the sections to dry out as this produces staining artifacts. Use a humidified chamber for all incubations.
  • Always use freshly prepared buffers. Bacterial contamination may affect the quality of the staining.
  • All tissues should be well fixed. Fixation should be sufficient to maintain the integrity of the tissue, but not so harsh as to destroy the antigen under study.
  • Sections which are thicker than the recommended 4 microns may require longer incubation times for optimal staining.

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Preparation of Modifed Radioimmunoprecipitation (RIPA) Buffer

Activation of Sodium Orthovanadate

Preparation of Cell Lysate

Western Immunoblotting of Proteins

Troubleshooting Tips for Western Immunoblotting

Immunoprecipitation of Proteins

Immunoprecipitation / Kinase Assay

Troubleshooting Tips for Immunoprecipitation / Kinase Assays

Assay for Immunoprecipitated Phosphoinositide 3-Kinase (PI 3 Kinase) Activity (in vitro)

Phospholipid Vesicle Preparation

Chromatin Immunoprecipitation (ChIPs) Assay Kit

Forskolin Treatment of Cells

Preparation of Brain Cytosol

Immunohistochemistry with Fixed Paraffin-Embedded Tissue Sections

Tips for Immunohistochemistry with Fixed Paraffin-Embedded Tissues

Immunohistochemical Methods to Detect Nitrotyrosine

Immunocytochemistry with Adherent Cells

Differentiation of Stages in the Cell Cycle

Elution of GST-Fusion Protein from Glutathione Agarose

Purification of Antiserum or Ascites by Protein A/G Chromatography

Molecular Weights of Some Common Proteins

Transient Transfection of Cos-1 Cells

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