Cells were seeded on chamber slides (BD, Heidelberg, Germany). After 24 hours (h), immunostaining was performed as published previously [16 (link)]. Briefly, after 1 min fixation with 4% paraformaldehyde (PFA), cells were washed with PBS and incubated in PBS containing 5% bovine serum albumin (BSA). Then, 0.1% Tween/PBS (30 s) was applied, followed by immunostaining. Anti-human PROX1 antibody (1:200, ReliaTech, Wolfenbüttel, Germany), and anti-human CD31 antibody (1:50; BD, Franklin Lakes, NJ, USA) were incubated for 1h, cells were rinsed with PBS, and secondary antibody staining with Alexa594-conjugated goat-anti-rabbit and Alexa488-conjugated goat-anti-mouse antibodies (1:200; Thermo Fisher Scientific, Waltham, MA, USA) was performed. Antibodies were diluted in PBS containing 5% BSA. Cells were counterstained with DAPI and analyzed with AxioImager Z.1 (Zeiss, Göttingen, Germany) and processed with AdobePhotoshop (Adobe, San Jose, CA, USA).
Alexa594 conjugated goat anti rabbit
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa594-conjugated goat-anti-rabbit is a secondary antibody conjugated with the fluorescent dye Alexa Fluor 594. It is designed to detect and label rabbit primary antibodies in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager z1, by Zeiss (2 mentions)
Chamber slide, by BD (2 mentions)
Alexa 488 conjugated goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Anti human cd31 antibody, by BD (1 mentions)
Mouse anti human vimentin, by Agilent Technologies (1 mentions)
Anti human cd31, by BD (1 mentions)
Carl confocal imaging system lsm 780, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Mouse anti flag, by Merck Group (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
4 protocols using alexa594 conjugated goat anti rabbit
1
Immunostaining for PROX1 and CD31
2
Immunostaining of PROX1, CD31, Vimentin, and αSMA
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Cells were seeded on chamber slides (BD). After 24 hours, immunostaining was performed as follows: after short (1 min) 4% paraformaldehyde (PFA) fixation, cells were washed with PBS and incubated in bovine serum albumin (BSA). Then 0.1% Tween/PBS (for better PROX1 staining results—30 seconds) was applied, followed by immunostaining. Anti-human PROX1 (1:200, ReliaTech, Wolfenbüttel, Germany), anti-human CD31 (1:50; BD, NJ, USA), mouse-anti-human vimentin (1:1, DAKO; clone V9), and mouse-anti-human smooth muscle α-actin (αSMA) (1:500, Sigma, clone 1A4) antibodies were incubated for one hour, rinsed with PBS, and then secondary antibody staining with Alexa594-conjugated goat-anti-rabbit and Alexa488-conjugated goat-anti-mouse (1:200; ThermoFisher Scientific, MA, USA) was performed. Cells were counter-stained with Dapi and analyzed with AxioImager Z.1 (Zeiss, Göttingen, Germany).
3
Immunofluorescence Detection of SMAD2/3
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To detect the expression and translocation of SMAD2/3, HCT116 cells which were prepared in cover glass were fixed for 10 min by 4% PFA. The cell membrane was permeabilized with 0.5% Triton X-100 for 10 min. After washing with 0.1% Tween20 in PBS [phosphate buffered saline (PBST)], cells were incubated with blocking buffer (5% bovine serum albumin, and 0.1% Triton-X 100 in PBS) for 1 h at room temperature. The primary antibodies against SMAD2/3 (1:200 dilution, Cell Signaling, Danvers, Massachusetts, USA), were incubated with fixed cells at 4°C overnight with gentle agitation. After washing completely with PBST, Alexa 594 conjugated goat antirabbit (1:5000 dilution, Life Technologies, Carlsbad, California) antibodies were added into a blocking buffer for 1 h at room temperature in a dark chamber. Cell nuclei were stained with DAPI (Vector Laboratories, Inc.; Burlingame, California, USA) for 2 min at room temperature. Fluorescence images were taken by the Carl Zeiss Confocal Imaging System (LSM 780; Carl Zeiss, Jena, Germany).
4
Visualizing Toxoplasma gondii Invasion
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1.25×106T. gondii transfected with pU6-SAG1 were added to confluent monolayers of HFF cells grown on cover slips in 24-well dishes. Cells were fixed 24 hpi on ice with methanol for 2 minutes, then stained using mouse-anti-SAG1 (mAb DG52), rabbit-anti-TgAct1 [11] (link), mouse-anti-Ty (mAb BB2 [12] (link)), or mouse-anti-Flag (Sigma, F1804). Alexa-594-conjugated goat-anti-rabbit (Life Technologies, A11037) and Alexa-488-conjugated goat-anti-mouse (Life Technologies, A11029) were used as secondary antibodies. Antibodies against Toxoplasma were kindly provided by L. D. Sibley (Washington University School of Medicine, USA). Coverslips were mounted in Prolong Gold containing DAPI (Invitrogen P-36931) and imaged using an Eclipse Ti microscope (Nikon).
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