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GAD1 (A9A5X) Rabbit mAb
Primary Antibodies

Monoclonal Antibody - GAD1 (A9A5X) Rabbit mAb - Immunofluorescence (Frozen), UniProt ID Q99259, Entrez ID 2571 #63080


R Endogenous Rabbit IgG
IF-F - GAD1 (A9A5X) Rabbit mAb

Confocal immunofluorescent analysis of rat cerebellum at low (left) and high magnification (right) using GAD1 (A9A5X) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IF-F - GAD1 (A9A5X) Rabbit mAb

Confocal immunofluorescent analysis of rat retina (left) and pancreas (right) using GAD1 (A9A5X) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Immunofluorescence (Frozen)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer: (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) to 9.5 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

    NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Frozen/Cryostat Sections (IF-F)

  1. For fixed frozen tissue proceed with Immunostaining (Section C).
  2. For fresh, unfixed frozen tissue, please fix immediately, as follows:
    1. Cover sections with 4% formaldehyde dilute in 1X PBS.

      NOTE: Formaldehyde is toxic, use only in fume hood.

    2. Allow sections to fix for 15 minutes at room temperature.
    3. Aspirate liquid, rinse three times in 1X PBS for 5 minutes each.
    4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised July 2016

Protocol Id: 151

Application Dilutions
Immunofluorescence (Frozen) 1:100

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

GAD1 (A9A5X) Rabbit mAb recognizes endogenous levels of total GAD1 protein. This antibody cross-reacts with serum albumin.

Species Reactivity:


Species predicted to react based on 100% sequence homology:

Human, Mouse

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala15 of human GAD1 protein.

The enzyme glutamate decarboxylase (GAD) is responsible for the synthesis of the essential neurotransmitter gamma-aminobutyric acid (GABA) from L-glutamic acid (1). GAD1 (GAD67) and GAD2 (GAD65) are expressed in nervous and endocrine systems (2) and are thought to be involved in synaptic transmission (3) and insulin secretion (4), respectively. Autoantibodies against GAD2 may serve as markers for type I diabetes (5). Many individuals suffering from an adult onset disorder known as Stiff Person Syndrome (SPS) also express autoantibodies to GAD2 (6).

GAD1 and GAD2 are encoded by separate genes and are coexpressed in most of the GABA-containing neurons (1, 7).

  1. Kaufman, D.L. et al. (1991) J Neurochem 56, 720-3.
  2. Feldblum, S. et al. (1993) J Neurosci Res 34, 689-706.
  3. Gao, B. and Moore, R.Y. (1996) J Biol Rhythms 11, 172-9.
  4. Rubi, B. et al. (2001) J Biol Chem 276, 36391-6.
  5. Gilliam, L.K. et al. (2004) Clin Exp Immunol 138, 337-41.
  6. Skorstad, G. et al. (2008) Eur J Neurol 15, 973-80.
  7. Erlander, M.G. et al. (1991) Neuron 7, 91-100.
Entrez-Gene Id
Swiss-Prot Acc.
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.

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