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94242
GFAP (E4L7M) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate)
Antibody Conjugates
Monoclonal Antibody
R
Recombinant

GFAP (E4L7M) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #94242

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Filter:
  1. IHC
  2. IF
Immunohistochemical analysis of paraffin-embedded mouse brain using GFAP (E4L7M) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (green) and DAPI #4083 (blue).
Confocal immunofluorescent analysis of fixed frozen mouse hippocampus labeled with GFAP (E4L7M) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (green), α-Synuclein (E4U2F) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #33611 (red), and ProLong Gold Antifade Reagent with DAPI #8961 (blue).
Confocal immunofluorescent analysis of U-251 MG cells (left, positive) and HeLa cells (right, negative) using GFAP (E4L7M) XP® Rabbit mAb (Alexa Fluor 488® Conjugate) (green), Dylight 554 Phalloidin #13054 (red), and DAPI #4083 (blue).
To Purchase # 94242
Cat. # Size Qty. Price
94242S
100 µl  (50 tests)

Supporting Data

REACTIVITY H M R Mk
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Rabbit IgG

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Description

This Cell Signaling Technology® antibody is conjugated to Alexa Fluor® 488 fluorescent dye under optimal conditions. This antibody conjugate is expected to exhibit the same species cross-reactivity as the unconjugated GFAP (E4L7M) XP® Rabbit mAb #80788.

Product Usage Information

Application Dilution
Immunohistochemistry (Paraffin) 1:50
Immunofluorescence (Frozen) 1:50 - 1:200
Immunofluorescence (Immunocytochemistry) 1:100

Storage

Supplied in PBS (pH 7.2), less than 0.1% sodium azide, and 2 mg/mL BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Protocol

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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

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

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1 L 1X TBST, add 100 ml 10X Tris Buffered Saline with Tween® 20 (TBST)(#9997) to 900 ml dH20, mix.
    2. 1X Phosphate Buffered Saline (PBS): To prepare 1L 1X PBS, add 100 mL 10X Wash Buffer, Phosphate Buffered Saline(#12528) to 900 ml dH20, mix.
  5. SignalStain® Antibody Diluent: (#8112)
  6. 1X EDTA Unmasking Solution: To prepare 250 mL of 1X EDTA unmasking solution, dilute 25 ml of SignalStain® EDTA Unmasking Solution (10X) (#14747) with 225 ml of dH2O.
  7. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  8. Blocking Solution: 1X TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O, add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  9. Prolong® Gold AntiFade Reagent(#9071), Prolong® Gold AntiFade Reagent with DAPI(#8961).
  10. (optional) TrueBlack® Lipofuscin Autofluorescence Quencher (#92401).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 minutes each.
    2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
    3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
  2. Wash sections twice in dH2O for 5 minutes each.

C. Antigen Unmasking

  1. Heat slides submersed in 1X EDTA unmasking solution in a microwave until boiling is initiated; follow with 15 min at sub-boiling temperature (95°-98°). No cooling is necessary.
  2. D. Staining

    1. Wash sections in dH2O three times for 5 minutes each.
    2. Incubate sections in 1X TBST for 5 min.
    3. Block each section with 100-400 µl of preferred blocking solution for 1 hour at room temperature.
    4. Remove blocking solution and add 100-400 µl primary antibody diluted in SignalStain® Antibody Diluent to each section.
    5. Incubate overnight at 4°C.
    6. Rinse three times in 1X PBS for 5 min each protected from light.
      NOTE: See below for optional TrueBlack® Lipofuscin Autofluorescence Quencher protocol.
    7. Coverslip slides with Prolong® Gold Antifade Reagent or Prolong® Gold Antifade Reagent with DAPI.
    8. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

    TrueBlack® Lipofuscin Autofluorescence Quencher protocol

    Following Section D Step 6:

    IMPORTANT: TrueBlack® Lipofuscin Autofluorescence Quencher is not compatible with detergent. Any steps involving detergent must be done before applying TrueBlack® Lipofuscin Autofluorescence Quencher.

    1. Prepare TrueBlack® Lipofuscin Autofluorescence Quencher solution by diluting 1:20 in 70% ethanol. Vortex to mix.
      NOTE: Quenching solution should be made fresh prior to use and discarded if precipitate is visible. We recommend heating the vial of stock solution of TrueBlack® Lipofuscin Autofluorescence Quencher, 20X in DMF to 70°C prior to dilution in order to avoid precipitate formation.
    2. Immediately cover tissue sections with 100 µL - 200 µL of quenching solution for 30 seconds at room temperature.
      IMPORTANT: Do not allow sections to dry out. Sections may tolerate longer incubations (up to 3 minutes) so long as they remain hydrated.
    3. Tap slides on an absorbent towel to collect excess TrueBlack® Lipofuscin Autofluorescence Quencher before transferring to 1X PBS.
    4. Rinse three times in 1X PBS for 5 min each.
    5. Proceed with counterstaining/mounting.

Protocol Id: 2924

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 goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. 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. 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 protocol on product webpage 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. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. 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 November 2013

Protocol Id: 222

Immunofluorescence (Immunocytochemistry)

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 goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. 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. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

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

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min 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 product webpage 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. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. 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 November 2013

Protocol Id: 182

Specificity / Sensitivity

GFAP (E4L7M) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total GFAP protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to human GFAP protein.

Background

The cytoskeleton consists of three types of cytosolic fibers: microfilaments (actin filaments), intermediate filaments, and microtubules. Major types of intermediate filaments are specifically expressed in particular cell types: cytokeratins in epithelial cells, glial fibrillary acidic protein (GFAP) in glial cells, desmin in skeletal, visceral, and certain vascular smooth muscle cells, vimentin in cells of mesenchymal origin, and neurofilaments in neurons. GFAP and vimentin form intermediate filaments in astroglial cells and modulate their motility and shape (1). In particular, vimentin filaments are present at early developmental stages, while GFAP filaments are characteristic of differentiated and mature brain astrocytes. Thus, GFAP is commonly used as a marker for intracranial and intraspinal tumors arising from astrocytes (2). In addition, GFAP intermediate filaments are also present in nonmyelin-forming Schwann cells in the peripheral nervous system (3).

Limited Uses

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For Research Use Only. Not for Use in Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
XP is a registered trademark of Cell Signaling Technology, Inc.
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or [email protected].
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