Caco-2BBe monolayers grown on 5 cm2 Transwell supports were fixed in 2% paraformaldehyde and immunostained as described previously. Snap-frozen tissues were stored at −80°C, after which 5 μm frozen sections were cut and fixed in 1% paraformaldehyde. EdU was detected using the Alexa Fluor 488 click chemistry detection kit (Invitrogen). Immunostaining used affinity-purified rabbit anti-human MLCK1 (2 μg/ml),7 (link) monoclonal mouse anti-MLCK clone K36 (Sigma-Aldrich, 1 μg/ml)7 (link), affinity-purified rabbit anti-pMLC (Cell Signaling 3671, 1 μg/ml), monoclonal mouse anti-occludin clone OC-3F10 (Invitrogen, 1 μg/ml), monoclonal mouse anti-E-cadherin clone M168 (Abcam, 2 μg/ml), or monoclonal rat anti-CD3 clone CD3–12 (Abcam, 2 μg/ml). Primary antibodies were followed by Alexa Fluor 488- or Alexa Fluor 594-conjugated affinity-purified secondary antibodies (Invitrogen) along with Alexa Fluor 594-conjugated phalloidin (Invitrogen) and Hoechst 33342 (Invitrogen). Stained sections were mounted in Prolong gold (Invitrogen).
Rabbit anti pmlc
Manufactured by Cell Signaling Technology
Sourced in Germany
Rabbit anti-pMLC is a primary antibody that specifically recognizes the phosphorylated form of myosin light chain (MLC). This antibody can be used to detect and quantify the levels of phosphorylated MLC in various cell and tissue samples through techniques such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 conjugated phalloidin, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 click chemistry detection kit, by Thermo Fisher Scientific (2 mentions)
Rabbit anti human mlck1, by Merck Group (2 mentions)
Monoclonal mouse anti mlck clone k36, by Merck Group (2 mentions)
Monoclonal mouse anti occludin clone oc 3f10, by Thermo Fisher Scientific (2 mentions)
Monoclonal mouse anti e cadherin clone m168, by Abcam (2 mentions)
Monoclonal rat anti cd3 clone cd3 12, by Abcam (2 mentions)
Alexa fluor 488 or alexa fluor 594 conjugated affinity purified secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Schneider s drosophila medium, by Thermo Fisher Scientific (1 mentions)
Y27632, by Thermo Fisher Scientific (1 mentions)
Mouse anti ena, by Developmental Studies Hybridoma Bank (1 mentions)
Rat anti de cad, by Developmental Studies Hybridoma Bank (1 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (1 mentions)
Mouse anti dlg, by Developmental Studies Hybridoma Bank (1 mentions)
Ab16667, by Abcam (1 mentions)
G2654, by Abcam (1 mentions)
Mouse anti glucagon, by Merck Group (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
5 protocols using rabbit anti pmlc
1
Immunostaining of Caco-2 and Tissue Sections
2
Immunostaining of Caco-2 and Tissue Sections
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Caco-2BBe monolayers grown on 5 cm2 Transwell supports were fixed in 2% paraformaldehyde and immunostained as described previously. Snap-frozen tissues were stored at −80°C, after which 5 μm frozen sections were cut and fixed in 1% paraformaldehyde. EdU was detected using the Alexa Fluor 488 click chemistry detection kit (Invitrogen). Immunostaining used affinity-purified rabbit anti-human MLCK1 (2 μg/ml),7 (link) monoclonal mouse anti-MLCK clone K36 (Sigma-Aldrich, 1 μg/ml)7 (link), affinity-purified rabbit anti-pMLC (Cell Signaling 3671, 1 μg/ml), monoclonal mouse anti-occludin clone OC-3F10 (Invitrogen, 1 μg/ml), monoclonal mouse anti-E-cadherin clone M168 (Abcam, 2 μg/ml), or monoclonal rat anti-CD3 clone CD3–12 (Abcam, 2 μg/ml). Primary antibodies were followed by Alexa Fluor 488- or Alexa Fluor 594-conjugated affinity-purified secondary antibodies (Invitrogen) along with Alexa Fluor 594-conjugated phalloidin (Invitrogen) and Hoechst 33342 (Invitrogen). Stained sections were mounted in Prolong gold (Invitrogen).
3
Pupal Eye Immunostaining Protocol
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Pupal eyes or wing imaginal discs were fixed and stained following standard formaldehyde fixation and permeabilization/washes in PBS containing 0.3% Triton X-100. All pupal eyes were fixed ∼40 h APF unless otherwise specified. For phalloidin staining after Triton X-100 permeabilization, tissue was fixed first, incubated in PBS containing 0.3% Triton X-100 and 5% goat serum for 1 h, and then washed three times in PBS with 5% goat serum and stained with phalloidin in PBS with 5% goat serum for 30 min. The following antibodies were used: rat anti–DE-cad (1:10), mouse anti–α-Spec (1:50), mouse anti-Dlg (1:50), mouse anti-Ena (1:50; all from the Developmental Studies Hybridoma Bank), mouse anti-Myc (1:200, clone 9E10; Millipore Sigma), rabbit anti-pMLC (1:10; Cell Signaling Technologies), rabbit anti–PAR-3 (1:1,000; provided by A. Wodarz, Univesity of Cologne, Cologne, Germany; Wodarz et al., 1999 (link)). Alexa Fluor 568 phalloidin (1:50; Invitrogen) was used to visualize F-actin.
In experiments involving treatment of Rok inhibitor, pupal eyes were incubated with 1 mM Y27632 in Schneider’s Drosophila medium (GIBCO BRL, Life Technologies) for 1 h before fixation as described previously (Legoff et al., 2013 (link)).
In experiments involving treatment of Rok inhibitor, pupal eyes were incubated with 1 mM Y27632 in Schneider’s Drosophila medium (GIBCO BRL, Life Technologies) for 1 h before fixation as described previously (Legoff et al., 2013 (link)).
4
Comprehensive Immunostaining Protocol
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These antibodies were used in this study for immunohistochemistry; Primary antibodies were goat anti-WNT4 (R&D system, AF475, 1:100) (Fig. 1g ), rabbit anti-WNT4 (Bioss Antibodies, BS-6134R, 1:100)(Supplementary Fig. 3 ), guinea pig anti-Insulin (DAKO, A0564, 1:100), mouse anti-Glucagon (Sigma, G2654, 1:800), chicken anti-GFP (Abcam, ab13970, 1:1000), rabbit anti-Ki67 (Abcam, ab16667, 1:100), mouse anti-active beta-catenin (Millipore, 05-665, 1:100), rabbit anti-pMLC (Cell Signaling Technology, 3674, 1:100). Secondary antibodies were donkey anti-mouse Alexa FluorTM 568 (Thermo Fisher, A10037, 1:1000), donkey anti-mouse Alexa Fluor® 647 (Jackson ImmunoResearch Europe Ltd, 715-605-150, 1:800), donkey anti-chicken Alexa Fluor® 488 (Jackson ImmunoResearch Europe Ltd, 703-545-155, 1:800), goat anti-chicken Alexa FluorTM 488 (Thermo Fisher, A-11039, 1:1000), donkey anti-goat Alexa Fluor® 488 (Abcam, ab150129, 1:1000), donkey anti-guinea pig Texas red (Abcam, ab6906, 1:300), donkey anti-guinea pig Biotin (Jackson ImmunoResearch Europe Ltd, 706-065-1480, 1:400), donkey anti-rabbit Biotin (Jackson ImmunoResearch Europe Ltd, 711-065-152, 1:200), Streptavidin Alexa Fluor® 647 (Jackson ImmunoResearch Europe Ltd, 016-600-084, 1:1000). Nuclei were stained with DAPI (Sigma, D9542-1MG, 1:10000).
5
GEF-H1 Regulation in Cytoskeleton Dynamics
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fMLP, blebbistatin, anti–human IgG F(ab')2, and recombinant human TNF were obtained from Sigma-Aldrich. C5a and ICAM-1/Fc chimera were from R&D Systems. Nocodazole and Y-27632 were obtained from EMD Millipore. Latrunculin A was obtained from Alexis Corporation.
Antibodies used for immunoblotting were mouse anti-MLC (1:2,000; Sigma-Aldrich), rabbit anti-pMLC (1:500; Cell Signaling Technology), sheep anti–GEF-H1 (1:500; Exalpha Biologicals), and mouse anti-GAPDH (1:26,000; Sigma-Aldrich).
Antibodies used for immunofluorescent staining were mouse anti–GEF-H1 (1:50; Hycult), rabbit anti–GEF-H1-pS885 (1:200; Cell Signaling Technology), rabbit anti–flotillin-2 (1:100; Santa Cruz Biotechnology, Inc.), rat anti–α-tubulin (1:100; Abcam), and mouse anti-pMLC (1:50; Cell Signaling Technology).
Antibodies used for immunoblotting were mouse anti-MLC (1:2,000; Sigma-Aldrich), rabbit anti-pMLC (1:500; Cell Signaling Technology), sheep anti–GEF-H1 (1:500; Exalpha Biologicals), and mouse anti-GAPDH (1:26,000; Sigma-Aldrich).
Antibodies used for immunofluorescent staining were mouse anti–GEF-H1 (1:50; Hycult), rabbit anti–GEF-H1-pS885 (1:200; Cell Signaling Technology), rabbit anti–flotillin-2 (1:100; Santa Cruz Biotechnology, Inc.), rat anti–α-tubulin (1:100; Abcam), and mouse anti-pMLC (1:50; Cell Signaling Technology).
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