Testing and titration of primary antibody

NDT401d

Testing & titration of primary antibody for colorimetric immunostaining on free-floating sections (NDT401d-a):

This service includes tissue preparation, sectioning, immunolabeling with various dilutions of the primary antibody, mounting, coverslipping and labeling the slides. As a result, you will receive up to 30 immunostained tissue sections ready for microscopic observations. In addition, the experimental procedure used for the antibody titration will be provided.

Procedure: Following cryoprotection, tissue will be rapidly frozen in isopentane pre-cooled to -70°C. The frozen tissue will then be cut on a cryostat and collected in our unique section cryoprotection solutions (NDT301). Subsequently, sections will be processed free-floating for immunostaining with the specific antibody at various dilutions according to the avidin-biotin-complex (ABC) method¹ (for see samples below)

Heat shock protein-immunoreactivity. This 30 µm cryostat section was cut from the dorsal hippocampus of a rat that survived for 15 hrs after the injection of aminooxyacetic acid into the entorhinal cortex (for details, cf. Neurosci. Lett. 147:185-188, 1992). The section was processed free-floating according to avidin-biotin-complex method.

Heat shock protein-immunostained section counterstained with thionin. This 30 µm cryostat section was cut from the dorsal hippocampus of a rat that survived for 15 hrs after the injection of aminooxyacetic acid into the entorhinal cortex (for details, cf. Neurosci. Lett. 147:185-188, 1992). The section was processed for heat shock protein-immunostaining (brown) and then counterstained with thionin.

OX18-immunostained section counterstained with thionin. This 30 µm cryostat section was cut from the entorhinal cortex of a rat that survived for 24 hrs after the injection of aminooxyacetic acid into the entorhinal cortex. The section was processed for OX-immunostaining and then counterstained with thionin. Note the expression of OX-18 immunoreactivity (brown).

GFAP-immunostained section counterstained with thionin. This 30 µm cryostat section was cut from the entorhinal cortex of a normal rat. The section was processed for GFAP-immunostaining and then counterstained with FD thionin solution (cf. Products, Cat. #PS101). Note GFAP-immunoreactivity (brown) in the processes of astrocytes.

Parvalbumin-immunostained section counterstained with thionin. 30 µm cryostat section through the medial entorhinal cortex of a rat that survived for 24 hrs after kainic acid administration, showing the preferential loss of neurons in layer III and relative resistance of parvalbumin neurons (for details, cf. J. Neurosci. 15:6301-6313, 1995).

Remarks:

  • A quotation is required before placing an order.
  • The investigator needs to provide both tissue and the specific antibody.

Reference:

  1. Hsu S. M., Raine L. and Fanger H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577-580.

Testing & titration of primary antibody for colorimetric immunostaining on sections mounted on slides (NDT401db):

This service includes tissue preparation, sectioning, immunolabeling with various dilutions of the primary antibody, coverslipping and labeling the slides. As a result, you will receive up to 30 immunostained tissue sections ready for microscopic observations. In addition, the experimental procedure used for the antibody titration will be provided.

Procedure: Following cryopretection, tissue will be rapidly frozen in isopentane pre-cooled to -70°C. The frozen tissue will then be cut on a cryostat and mounted on gelatin-coated microscope slides (Slides available upon request). Subsequently, sections will be processed on slides for immunostaining with the specific antibody at various dilutions according to the avidin-biotin-complex (ABC) method¹ (for psee samples below).

Cytokeratin 18-immunostained section counterstained with cresyl violet. This 12 µm frozen section of the rat prostate was processed for Cytokeratin 18-immunoreactivity (brown) and was then counterstained with FD cresyl violet solution (cf. Products, Cat. #NDT202)

CD31-immunostained section counterstained with hematoxylin. This 7 µm paraffin section of the mouse ear was processed for CD31-immunoreactivity (brown) and was then counterstained with FD hematoxylin solution (cf. Products, Cat. #NDT204).

Remarks:

  • A quotation is required before placing an order.
  • The investigator needs to provide both tissue and the specific antibody.

Reference:

  1. Hsu S. M., Raine L. and Fanger H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577-580.

Testing & titration of primary antibody for immunofluorescence labeling on free-floating sections (NDT401d-c):

This service includes tissue preparation, sectioning, immunolabeling with various dilutions of the primary antibody, mounting, coverslipping and labeling the slides. As a result, you will receive up to 30 immunostained tissue sections ready for microscopic observations. In addition, the experimental procedure used for the antibody titration will be provided.

Procedure: Following cryoprotection, tissue will be rapidly frozen in isopentane pre-cooled to -70°C. The frozen tissue will then be cut on a cryostat and collected in our unique section cryoprotection solution (NDT301). Subsequently, sections will be processed free-floating for immunostaining with the specific antibody at various dilutions according to the indirect immunofluorescence method¹ (for see samples below).

Cofocal image of BrdU-immunoreactivity. 30 µm cryostat section was cut from the hippocampal dentate gyrus of a mouse that survived for 24 hrs after the injection with 5-bromo-2-deoxyuridine (BrdU). This section was processed free-floating according to the indirect fluorescence method.

Cofocal image of NeuN-immunoreactivity. The same section as shown above was processed free-floating for NeuN-immuoreactivity according to the indirect fluorescence method. Note NeuN-labeled granule cells and polymorphic neurons in the dentate gyrus.

Colocalization of BrdU- and NeuN- immunoreactivities. A digital overlay of the 2 images shown above. Note that the regions of colocalization, reflecting the additive effect of superimposed green and red pixels, appear in yellow.

Remarks:

  • A quotation is required before placing an order.
  • The investigator needs to provide both tissue and the specific antibody.

Reference:

  1. Coons, A.H. (1958) Fluorescent antibody methods. In J.F. Danielli (ed): General Cytochemical Methods. New York: Academic Press, pp. 399-422.

Testing & titration of primary antibody for immunofluorescence labeling on sections mounted on slides (NDT401d-d):

This service includes tissue preparation, sectioning, immunolabeling with various dilutions of the primary antibody, coverslipping and labeling the slides. As a result, you will receive up to 30 immunostained tissue sections ready for microscopic observations. In addition, the experimental procedure used for the antibody titration will be provided.

Procedure: Following cryoprotection, tissue will be rapidly frozen in isopentane pre-cooled to -70°C. The frozen tissue will then be cut on a cryostat and mounted on gelatin-coated microscope slides (Slides available upon request). Subsequently, sections will be processed on slides for immunostaining with the specific antibody at various dilutions according to the indirect immunofluorescence method¹. (for see samples below).

Substance P-immunofluorescence in the spinal cord. 10 µm cryostat section was cut transversely from the chicken spinal cord. This section was processed on slide according to the indirect fluorescence method (for details, cf. J. Comp. Neurol. 278:253-264, 1988). Note substance P containing fibers mainly in the dorsolateral funiculus, Lissauer’s tract and the dorsal horn.

Substance P-immunofluorescence in the spinal cord. A 10 µm cryostat section of the chicken spinal cord was processed as described above. Note dense substance P immunoreactivity in the dorsolateral funiculus and the dorsal horn.

Vasoactive intestinal polypeptide-immunofluo-rescence in the spinal cord. A 10 µm cryostat section of the chicken spinal cord was processed on slide according to the indirect fluorescence method. Note 2 large neurons containing vasoactive intestinal polypeptide in the nucleus of the dorsolateral funiculus (for details, cf. J. Comp. Neurol. 278:253-264, 1988).

Remarks:

  • A quotation is required before placing an order.
  • The investigator needs to provide both tissue and the specific antibody.

Reference:

  1. Coons, A.H. (1958) Fluorescent antibody methods. In J.F. Danielli (ed): General Cytochemical Methods. New York: Academic Press, pp. 399-422.

Testing & titration of primary antibody for immunogold labeling on free-floating sections (NDT401d-e):

This service includes tissue preparation, sectioning, immunolabeling, mounting, coverslipping and labeling the slides. As a result, you will receive up to 60 immunolabeled sections per brain or per tissue block ready for microscopic observations.

Procedure: Following cryoprotection, tissue will be rapidly frozen in isopentane pre-cooled to -70°C. The frozen tissue will then be cut on a cryostat and collected in our unique section cryoprotection solution (NDT301). Subsequently, the sections cut from various levels (or the levels of your choice) will be processed free-floating for immunostaining with 1 specific antibody according to immunogold labeling technique¹ (cf. see samples below).

Tyrosine hydroxylase-immunoreactivity in the rat substantia nigra. 30 µm cryostat section cut coronally from the midbrain of a normal rat. This section was processed free-floating according to the immunogold labeling technique. Note densely labeled neurons in the compact area of the substantia nigra.

Tyrosine hydroxylase-immunoreactivity in the rat substantia nigra. The high magnification of the substantia nigra as shown above. Note that dense black deposits in the cytoplasm of neurons are actually metallic silver grains, which can also be viewed under a microscope with a darkfield condenser.

Remarks:

  • A quotation is required before placing an order.
  • The investigator needs to provide both tissue and the specific antibody.

Reference:

  1. Humbel B. M., Sibon O. C. M., Stierhof Y.-D. and Schwarz H. (1995) Ultra-small gold particles and silver enhancement as a detection system in immunolabeling and In Situ Hybridization experiments. J. Histochem. Cytochem. 43, 735-737.

Testing & titration of primary antibody for immunogold labeling on sections mounted on slides (NDT401d-f):

This service includes tissue preparation, sectioning, immunolabeling with various dilutions of the primary antibody, coverslipping and labeling the slides. As a result, you will receive up to 30 immunostained tissue sections ready for microscopic observations. In addition, the experimental procedure used for the antibody titration will be provided.

Procedure: Following cryoprotection, tissue will be rapidly frozen in isopentane pre-cooled to -70°C. The frozen tissue will then be cut on a cryostat and mounted on gelatin-coated microscope slides (Slides available upon request). Subsequently, sections will be processed on slides for immunostaining with the specific antibody at various dilutions according to the immunogold labeling technique¹.

Remarks:

  • A quotation is required before placing an order.
  • The investigator needs to provide both tissue and the specific antibody.

Reference:

  1. Humbel B. M., Sibon O. C. M., Stierhof Y.-D. and Schwarz H. (1995) Ultra-small gold particles and silver enhancement as a detection system in immunolabeling and In Situ Hybridization experiments. J. Histochem. Cytochem. 43, 735-737.


Terms and Conditions

  • For quality assurance of our service, it is recommended that you discuss with us for preferred perfusion protocol and histology and/or immunolabeling protocols.
  • It is suggested that you use Gel-coated microscopic slides for tissue mounting and 0.17um-thick coverslips.
  • A 15% of the fee will be due upon authorization of the study; and the remaining fee will be due upon delivery of study results.
  • Progress of the service is contingent upon staining quality of tissues, operated by the independent contractor.
  • Should early termination occur, Neurodigitech will prorate the cost incurred and invoice the Sponsor. The first portion of the fee is non-refundable.