Tissue sections (40 μm) and cultured cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. The samples were then blocked with 10% donkey serum and probed with anti-CD31 (RD, AF3628), anti-Laminin (Abcam, ab11575), anti-VE-cadherin (Santa Cruz, sc-9989), anti-occludin (Proteintech, 66,378-1-Ig), anti-claudin 5 (Thermo, 35-2500), anti-NRF1 (CST, 46743S), anti-CAV-1 (CST, 3267), anti-ZO-1 (Proteintech, 21,773-1-AP) or anti-LC3 (CST, 12741S) antibodies. The binding of primary antibodies was visualized with either Alexa Fluor 555-conjugated donkey anti-rabbit IgG (Thermo, A31572), Alexa Fluor 647-conjugated donkey anti-mouse IgG (Thermo, A32787), or Alexa Fluor 488-conjugated donkey anti-goat IgG (Abcam, ab150133). The samples were then counterstained with DAPI (Thermo) and imaged using a Leica SP8 confocal microscope.
Alexa fluor 647 conjugated donkey anti mouse igg
Manufactured by Thermo Fisher Scientific
Alexa Fluor 647-conjugated donkey anti-mouse IgG is a secondary antibody used for the detection and quantification of mouse primary antibodies in various immunoassays. The antibody is conjugated with the fluorescent dye Alexa Fluor 647, which has an excitation maximum at 650 nm and an emission maximum at 665 nm.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Anti claudin 5, by Thermo Fisher Scientific (1 mentions)
Anti cd31, by R&D Systems (1 mentions)
Alexa fluor 555 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti laminin, by Abcam (1 mentions)
Alexa fluor 488 conjugated donkey anti goat igg, by Abcam (1 mentions)
Anti zo 1, by Proteintech (1 mentions)
Anti occludin, by Proteintech (1 mentions)
Anti ve cadherin, by Santa Cruz Biotechnology (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Alexa fluor 594 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
A0855, by Merck Group (1 mentions)
Cellulase onozuka r 10, by Duchefa Biochemie (1 mentions)
3 protocols using alexa fluor 647 conjugated donkey anti mouse igg
1
Immunostaining of Endothelial Cell Markers
2
Immunofluorescence Staining Antibodies Protocol
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We used a mouse monoclonal antibody to β-Actin (clone AC-74; Sigma-Aldrich), a mouse monoclonal antibody to Giantin (clone G1/133; Enzo Life Sciences), a rabbit polyclonal antibody to GOLPH3 (Abcam, cat # ab98023), and a sheep polyclonal antibody to TGN46 (AbD Serotec, cat # AHP500G). We also used a homemade, mouse polyclonal antibody to human GOLPH3 that was described before [34 (link)], and a rabbit serum to GFP that was also described elsewhere [35 ]. HRP–conjugated secondary antibodies were from Jackson ImmunoResearch. The following fluorochrome-conjugated antibodies were from ThermoFisher: Alexa Fluor-594–conjugated donkey anti-rabbit IgG, Alexa Fluor-647–conjugated donkey anti-mouse IgG, Alexa Fluor-350–conjugated donkey anti-sheep IgG and Alexa Fluor-647–conjugated donkey anti-sheep IgG. Primary antibodies were used at a dilution 1/200 to 1/2000. HRP–or Alexa Fluor–conjugated secondary antibodies were used at dilutions 1/1000 to 1/20000, depending on their reactivity. The fluorescent nuclear stain 4’,6-diamidino-2-phenylindole (DAPI) was also from ThermoFisher.
3
Immunolocalization of Auxin in Plant Tissues
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The basal region of shoot explants was manually sectioned with a razor blade and immediately fixed in freshly prepared 3% 1-ethyl-3-(dimethylaminopropyl)-carbodiimide hydrochloride (EDAC) (Sigma-Aldrich) in 1× PBS containing 0.1% Triton X-100 (PBS-T) for 1 h on ice.
The sample sections were post-fixed for 1-2 h in 4% paraformaldehyde in PBS-T and washed three times in PBS-T for 5 min. The sample sections were then dehydrated and rehydrated with 30%, 50%, 70%, 100%, 70%, 50%, and 30% methanol in PBS-T (5 min each step) and incubated with 2% cellulase Onozuka R-10 (Duchefa Biochemie) for 30 min at 25 °C. After washing as above, sections were incubated overnight at 4 °C with a 1:100 dilution of the anti-IAA-C-monoclonal antibody (A0855; Sigma-Aldrich) in PBS-T. The sections were then rinsed three times for 10 min with PBS-T. Samples were incubated with 1:200 dilution of Alexa Fluor 647-conjugated donkey antimouse IgG (ThermoFisher Scientific) antibody for 2 h at 25 °C. The prepared samples were observed with LCSM as described elsewhere (Bustillo-Avendaño et al., 2018) .
The sample sections were post-fixed for 1-2 h in 4% paraformaldehyde in PBS-T and washed three times in PBS-T for 5 min. The sample sections were then dehydrated and rehydrated with 30%, 50%, 70%, 100%, 70%, 50%, and 30% methanol in PBS-T (5 min each step) and incubated with 2% cellulase Onozuka R-10 (Duchefa Biochemie) for 30 min at 25 °C. After washing as above, sections were incubated overnight at 4 °C with a 1:100 dilution of the anti-IAA-C-monoclonal antibody (A0855; Sigma-Aldrich) in PBS-T. The sections were then rinsed three times for 10 min with PBS-T. Samples were incubated with 1:200 dilution of Alexa Fluor 647-conjugated donkey antimouse IgG (ThermoFisher Scientific) antibody for 2 h at 25 °C. The prepared samples were observed with LCSM as described elsewhere (Bustillo-Avendaño et al., 2018) .
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