CST - p44/42 MAPK (Erk1/2) (L34F12) Mouse mAb

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REACTIVITY	SENSITIVITY	MW (kDa)	Isotype
H M R Mk Mi Z B Pg	Endogenous	42, 44	Mouse IgG1

Western blot analysis of extracts from NIH/3T3, PC12 and COS cells, using p44/42 MAP Kinase (L34F12) Mouse mAb.

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Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using p44/42 MAP Kinase (L34F12) Mouse mAb in the presence of control peptide (left) or p44/42 MAP Kinase Blocking Peptide (#4696 Specific) #1245 (right).

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Immunohistochemical analysis of paraffin-embedded human colon carcinoma, showing cytoplasmic and nuclear localization, using p44/42 MAP Kinase (L34F12) Mouse mAb.

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Image

Immunofluorescence (Immunocytochemistry)

Confocal immunofluorescent analysis of NIH/3T3 cells, either U0126-treated (#9903, 10 μM, 2 hour, left) or PDGF-treated (#9909, 100 ng/ml, 20 min, right), using p44/42 MAP Kinase (L34F12) Mouse mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

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Flow cytometric analysis of Jurkat cells, U0126-treated (blue) or PMA-treated (green), using p44/42 MAP Kinase (L34F12) Mouse mAb compared to a nonspecific negative control antibody (red).

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Protocol

Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween^® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.

A. Solutions and Reagents

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

20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix.
10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH₂O, mix.
1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH₂O.
10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH₂O, mix.
10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH₂O, mix.
10X Tris Buffered Saline with Tween^® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH₂O, mix.
Nonfat Dry Milk: (#9999).
Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
Wash Buffer: (#9997) 1X TBST.
Primary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 1X TBST and mix well.
Biotinylated Protein Ladder Detection Pack: (#7727).
Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
Secondary Antibody Conjugated to HRP: Anti-mouse IgG, HRP-linked Antibody (#7076).
Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

Treat cells by adding fresh media containing regulator for desired time.
Aspirate media from cultures; wash cells with 1X PBS; aspirate.
Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
Microcentrifuge for 5 min.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

NOTE: Loading of prestained molecular weight markers (#13953, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm²) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

(Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
Wash three times for 5 min each with 15 ml of TBST.
Incubate membrane with Anti-mouse IgG, HRP-linked Antibody (#7076 at 1:2000) and Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
Wash three times for 5 min each with 15 ml of TBST.
Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised November 2013

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

Wash Buffer: Tris Buffered Saline with Tween^® 20 (TBST-10X) (#9997)
Stripping Buffer: To prepare 100 ml, mix 6.25 ml of 1M Tris-HCl pH 6.8, 10 ml of 20% SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H₂0. Make buffer fresh just prior to use.

B. Protocol

After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.
Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation).
Wash membrane six times for 5 min each in TBST.
(Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO^® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
Wash membrane again four times for 5 min each in TBST.
The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

posted June 2005

revised June 2016

Protocol Id: 19

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

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

Xylene.
Ethanol, anhydrous denatured, histological grade (100% and 95%).
Deionized water (dH₂O).
Hematoxylin (optional).
Wash Buffer:
1. 1X Tris Buffered Saline with Tween^® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween^® 20 (#9997) to 900 ml dH₂0, mix.
SignalStain^® Antibody Diluent (#8112).
1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain^® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH₂O.
3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H₂O₂ to 90 ml dH₂O.
Blocking Solution: 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).
Detection System: SignalStain^® Boost IHC Detection Reagents (HRP, Mouse #8125).
Substrate: SignalStain^® DAB Substrate Kit (#8059).
Hematoxylin: Hematoxylin (#14166).
Mounting Medium: SignalStain^® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

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

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

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

Wash sections in dH₂O three times for 5 min each.
Incubate sections in 3% hydrogen peroxide for 10 min.
Wash sections in dH₂O two times for 5 min each.
Wash sections in wash buffer for 5 min.
Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain^® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
Equilibrate SignalStain^® Boost Detection Reagent (HRP, Mouse #8125) to room temperature.
Remove antibody solution and wash sections with wash buffer three times for 5 min each.
Cover section with 1–3 drops SignalStain^® Boost Detection Reagent (HRP, Mouse #8125) as needed. Incubate in a humidified chamber for 30 min at room temperature.
Wash sections three times with wash buffer for 5 min each.
Add 1 drop (30 µl) SignalStain^® DAB Chromogen Concentrate to 1 ml SignalStain^® DAB Diluent and mix well before use.
Apply 100–400 µl SignalStain^® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
Immerse slides in dH₂O.
If desired, counterstain sections with hematoxylin (#14166).
Wash sections in dH₂O two times for 5 min each.
Dehydrate sections:
1. Incubate sections in 95% ethanol two times for 10 sec each.
2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
3. Repeat in xylene, incubating sections two times for 10 sec each.
Mount sections with coverslips and mounting medium (#14177).

posted February 2010

revised March 2016

Protocol Id: 280

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

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.
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.
Methanol, 100%
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)) and 0.5 mL 20X PBS to 9.0 mL dH₂O, mix well. While stirring, add 30 µl Triton™ X-100.
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.
Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:
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 multiwell plates, chamber slides or on coverslips.

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 fume hood.
Allow cells to fix for 15 minutes at room temperature.
Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
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.

Methanol Permeabilization Step: Cover cells with ice-cold 100% methanol (use enough to cover completely to a depth of 3–5 mm, DO NOT LET DRY), incubate in methanol for 10 minutes at –20°C, rinse in 1X PBS for 5 minutes.
Block specimen in Blocking Buffer for 60 minutes.
While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
Aspirate blocking solution, apply diluted primary antibody.
Incubate overnight at 4°C.
Rinse three times in 1X PBS for 5 minutes each.
Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
Rinse in 1X PBS as in step 6.
Coverslip slides with Prolong^® Gold Antifade Reagent (#9071), Prolong^® Gold AntiFade Reagent with DAPI (#8961).
For best results examine specimens immediately using appropriate excitation wavelength. For long term storage, store slides flat at 4°C protected from light.

posted November 2006

revised December 2010

Protocol Id: 146

Flow Cytometry General Protocol

If using whole blood, please follow the Flow Cytometry Whole Blood Protocol.

A. Solutions and Reagents

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

20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
16% Formaldehyde (methanol free).
100% methanol.
Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
Recommended Anti-Mouse secondary antibodies::

B. Fixation

Collect cells by centrifugation and aspirate supernatant.
Resuspend cells in 0.5–1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
Fix for 10 min at 37°C.
Chill tubes on ice for 1 min.
For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).

C. Permeabilization

NOTE: This step is critical for many CST antibodies.

Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above.
Incubate 30 min on ice.
Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

Aliquot 0.5–1 x 10⁶ cells into each assay tube (by volume).
Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.
Resuspend cells in 100 μl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
Incubate for 1 hr at room temperature.
Wash by centrifugation in 2–3 ml incubation buffer.
Resuspend cells in fluorochrome-conjugated secondary antibody, diluted in incubation buffer at the recommended dilution.
Incubate for 30 min at room temperature.
Wash by centrifugation in 2–3 ml incubation buffer.
Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
Incubate for at least 30 min at room temperature.
Analyze cells in DNA staining solution on flow cytometer.

posted June 2005

Flow Cytometry Whole Blood Protocol

If using cell lines, please follow the Flow Cytometry General Protocol.

A. Solutions and Reagents

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

20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix.
16% Formaldehyde (methanol free).
Triton™ X-100: To prepare 50 ml of 0.1% Triton™ X-100 add 50 μl Triton™ X-100 to 50 ml 1 X PBS and mix well.
50% methanol.
Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
Secondary Antibodies:

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

Aliquot 100 μl fresh whole blood per assay tube.
OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
Add 65 μl of 10% formaldehyde to each tube.
Vortex briefly and let stand for 15 min at room temperature.
Add 1 ml of 0.1% Triton™ X-100 to each tube.
Vortex and let stand for 30 min at room temperature.
Add 1 ml incubation buffer.
Pellet cells by centrifugation and aspirate supernatant.
Repeat steps 7 and 8.
Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
Incubate at least 10 min on ice.
Proceed with staining or store cells at -20°C in 50% methanol.

C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.

Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
Incubate for 30–60 min at room temperature.
Wash by centrifugation in 2–3 ml incubation buffer.
Resuspend cells in fluorochrome-conjugated secondary antibody diluted in incubation buffer according to the manufacturer’s recommendations.
Incubate for 30 min at room temperature.
Wash by centrifugation in 2–3 ml incubation buffer.
Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1), 4–17.

posted November 2008

revised September 2013

Protocol Id: 406

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Product Usage Information

Application	Dilutions
Western Blotting	1:2000
Immunohistochemistry (Paraffin)	1:500
Immunofluorescence (Immunocytochemistry)	1:100
Flow Cytometry	1:200

Storage: 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.

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Specificity / Sensitivity

p44/42 MAP Kinase (L34F12) Mouse mAb detects endogenous levels of total p44/42 MAP kinase (Erk1/Erk2) protein. In some systems this antibody may recognize p42/Erk2 more readily than p44/Erk1. The antibody does not cross-react with JNK/SAPK or p38 MAP kinase.

Species Reactivity: Human, Mouse, Rat, Monkey, Mink, Zebrafish, Bovine, Pig

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Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the sequence of p42 MAP Kinase.

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Background

Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs, such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3), and research investigators consider it an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors, such as U0126 and PD98059.

1. Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.

2. Baccarini, M. (2005) FEBS Lett 579, 3271-7.

3. Meloche, S. and Pouysségur, J. (2007) Oncogene 26, 3227-39.

4. Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.

5. Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.

6. Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.

7. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.

8. Marais, R. et al. (1993) Cell 73, 381-93.

9. Kortenjann, M. et al. (1994) Mol Cell Biol 14, 4815-24.

10. Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.

Entrez-Gene Id 5595 , 5594

Swiss-Prot Acc. P27361 , P28482

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Data Sheets & Documentation

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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PhosphoSitePlus® Resource

p44/42 MAPK (Erk1/2) (L34F12) Mouse mAb #4696

APPLICATIONS

Gallery: p44/42 MAPK (Erk1/2) (L34F12) Mouse mAb #4696

Protocol

Western Blotting Protocol

A. Solutions and Reagents

B. Protein Blotting

A general protocol for sample preparation.

C. Membrane Blocking and Antibody Incubations

I. Membrane Blocking

II. Primary Antibody Incubation

D. Detection of Proteins

Directions for Use:

Western Blot Reprobing Protocol

A. Solutions and Reagents

B. Protocol

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

B. Deparaffinization/Rehydration

C. Antigen Unmasking

D. Staining

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

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

C. Immunostaining

Flow Cytometry General Protocol

A. Solutions and Reagents

B. Fixation

C. Permeabilization

D. Immunostaining

E. Optional DNA Dye

Flow Cytometry Whole Blood Protocol

A. Solutions and Reagents

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies

Product Usage Information

Specificity / Sensitivity

Source / Purification

Background

Data Sheets & Documentation

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