Images provided the Independent Validation Program (badge number 029646)Formalin-fixed and paraffin embedded human placenta labeled with Rabbit Anti-MEK1 Polyclonal Antibody (ABIN686482) at 1:250 overnight at room temperature followed by conjugation to secondary antibody.
Paraformaldehyde-fixed, paraffin embedded human colon cancer, Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30min, Antibody incubation with MEK1 Polyclonal Antibody, Unconjugated at 1:500 overnight at 4°C, followed by a conjugated secondary for 20 minutes and DAB staining.
Human Polyclonal MAP2K1 Primary Antibody for ICC, IF - ABIN4333486
(1 Publications):
Pénzváltó, Lánczky, Lénárt, Meggyesházi, Krenács, Szoboszlai, Denkert, Pete, Győrffy: MEK1 is associated with carboplatin resistance and is a prognostic biomarker in epithelial ovarian cancer. in BMC cancer 2014
(PubMed)
Dual specificity protein kinase which acts as an essential component of the MAP kinase signal transduction pathway. Binding of extracellular ligands such as growth factors, cytokines and hormones to their cell-surface receptors activates RAS and this initiates RAF1 activation. RAF1 then further activates the dual-specificity protein kinases MAP2K1/MEK1 and MAP2K2/MEK2. Both MAP2K1/MEK1 and MAP2K2/MEK2 function specifically in the MAPK/ERK cascade, and catalyze the concomitant phosphorylation of a threonine and a tyrosine residue in a Thr-Glu-Tyr sequence located in the extracellular signal-regulated kinases MAPK3/ERK1 and MAPK1/ERK2, leading to their activation and further transduction of the signal within the MAPK/ERK cascade. Depending on the cellular context, this pathway mediates diverse biological functions such as cell growth, adhesion, survival and differentiation, predominantly through the regulation of transcription, metabolism and cytoskeletal rearrangements. One target of the MAPK/ERK cascade is peroxisome proliferator-activated receptor gamma (PPARG), a nuclear receptor that promotes differentiation and apoptosis. MAP2K1/MEK1 has been shown to export PPARG from the nucleus. The MAPK/ERK cascade is also involved in the regulation of endosomal dynamics, including lysosome processing and endosome cycling through the perinuclear recycling compartment (PNRC), as well as in the fragmentation of the Golgi apparatus during mitosis.
Images provided the Independent Validation Program (badge number 029646)Formalin-fixe...
Paraformaldehyde-fixed, paraffin embedded human colon cancer, Antigen retrieval by bo...
Image provided by the Independent Validation Program (badge number 29760). Lane 1: He...
Images for product: anti-Mitogen-Activated Protein Kinase Kinase 1 (MAP2K1) (AA 2-35) antibody
Images provided the Independent Validation Program (badge number 029646)Formalin-fixed and paraffin embedded human placenta labeled with Rabbit Anti-MEK1 Polyclonal Antibody (ABIN686482) at 1:250 overnight at room temperature followed by conjugation to secondary antibody.
Paraformaldehyde-fixed, paraffin embedded human colon cancer, Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30min, Antibody incubation with MEK1 Polyclonal Antibody, Unconjugated at 1:500 overnight at 4°C, followed by a conjugated secondary for 20 minutes and DAB staining.
Image provided by the Independent Validation Program (badge number 29760). Lane 1: HeLa cell extract, Lane 2: NIH/3T3 cell extracts, Lane 3: c6/36 mosquito cell extract (non-reactivespecies) probed with Rabbit Anti-MEK1 Polyclonal Antibody, Unconjugated at 1:200 overnight at 4˚C. Followed by conjugation to secondary antibody at 1:20000 for 60 min at 26˚C.
Positive control: Human placenta tissue stained with antibody
Negative control: Human breast adipocytes tissue stained with antibody
Isotype control: Human placenta tissue stained with isotype control
Secondary only control: Human placenta tissue stained with secondary antibody only
Protocol
Immunohistochemistry was performed on a Ventana NEXes automated platform; instrument manufacturer specific reagents are italicized.
1. Slides were preheated in convection oven at 60°C for 30 min
2. Deparaffinization procedure: - 3 changes of Xylene, 5 min each - 3 changes of 100% Ethanol, 3 min each - 3 changes of 95% Ethanol, 3 min each - Rinsed in distilled water, 3 changes
3. Heat retrieval procedure - Slides retrieved in 10.0 mM Citrate, pH6.0 in a 1000W microwave oven (~100°C) for 15 min. - Slides were allowed to cool (in citrate) for 30 min. - Slides were washed x 3 in Distilled water
4. NEXes instrument procedure, iView DAB paraffin protocol (*abridged*): - Slide chamber warmed to 37°C
5. Slides rinsed with *reaction buffer* x3
6. *iView Inhibitor (H2O2)* applied and incubated for 4 min
7. Slides rinsed with *reaction buffer*
8. Antibody Application - Primary antibody diluted 1:250 in PBS (100 microliter applied/slide) - Ventana Isotype control applied neat - Slides Incubated overnight at room temperature (~12 hours ~25°C)
9. Slides rinsed with *reaction buffer* x3
10. *iView Biotinylated IgG* applied and incubated for 8 min
11. Slides rinsed with *reaction buffer*
14. *iView Streptavidin-Horseradish Peroxidase* applied and incubated for 8 min
15. Slides rinsed with *reaction buffer*
16. *iView DAB/H2O2* applied and incubated for 8 min
17. Slides rinsed with *reaction buffer*
18. *iView Copper* applied and incubated for 4 min
19. Slides rinsed with *reaction buffer*
20. Slides washed in Dawn Detergent/tap water
21. Counterstain Procedure - Hematoxylin (Leica 560 MX) 30 sec - Slides washed in tap water, 1 min - Decolorized (10% Acetic Acid in 70% ethanol), 1 min - Slides washed in tap water, 1 min - Bluing (Austin Clear Ammonia), 1 min - Slides washed in tap water, 1 min
22. Dehydration/coverslipping procedure: - 3 changes of 95% Ethanol, 3 min each - 3 changes of 100% Ethanol, 3 min each - 3 changes of Xylene, 5 min each - Mounted with Permount
23. Imaging: Leica SCN 400F Whole Slide Scanner with Digital Image Hub and Leica Slidepath software
Experimental Notes
Deviations from protocol/procedure supplied by manufacturer:
Step 1: Heated tissue 60°C for 30 minutes; manufacturer heats for 45 minutes.
Step 2: No ethanol wash was performed during deparaffinization; manufacturer includes 1 wash of 80% ethanol for 3 minutes.
Step 3.1: Slides were heated for 15 minutes; manufacturer provides a range of 15-20 minutes.
Step 3.2: Slides were cooled for 30 minutes; manufacturer cools for 20 minutes.
Step 4: Italicized reagents and incubation time are fixed instrument parameters.
Step 5: Secondary species-specific serum block not used; manufacturer blocks with 5% normal goat serum for 2 hours.
Step 8.1: Antibody diluted in PBS at 1:250; manufacture did not recommend diluent or dilution.
Step 8.2.1: Primary antibody incubated at room temperature overnight; manufacturer incubates overnight 4°C with agitation.
Images
Images
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Validation Images
Figure 1: Human placenta tissue stained with anti-MAP2K1 (brown) and counterstained w...
Figure 2: Human breast adipose tissue stained with anti-MAP2K1 (brown) and countersta...
Figure 3: Human placenta tissue stained with isotype control antibody (brown) and cou...
Figure 4: Human placenta tissue stained with secondary antibody only (brown) and coun...
Images for product:
Figure 1: Human placenta tissue stained with anti-MAP2K1 (brown) and counterstained with hematoxylin.
Figure 2: Human breast adipose tissue stained with anti-MAP2K1 (brown) and counterstained with hematoxylin.
Figure 3: Human placenta tissue stained with isotype control antibody (brown) and counterstained with hematoxylin.
Figure 4: Human placenta tissue stained with secondary antibody only (brown) and counterstained with hematoxylin.