Recombinant (human CCL1/ I-309; mouse TCA3), goat polyclonal antibody against CCL1, as well as IgG controls were obtained from R&D systems (Minneapolis, MN). Poxvirus MC148 was obtained from Abcam (Cambridge, MA). Anti-CCR8 was generated as described (10). Anti-CCR8 was conjugated using the APC Conjugation Kit (ab201807) which purchased from Abcam. Antibodies against MSC markers PDGFRα (clone αR1) and Purified Mouse Anti-Rat Nestin Clone Rat 401 (RUO) was purchased from BD Biosciences. One Step Staining Human Treg Flow Kit (FOXP3 Alexa Fluor 488/CD25 PE/CD4 PerCP) was purchased from Biolegend. Secondary antibodies Alexa Fluor 633 goat anti–rabbit IgG, Alexa Fluor 633 goat anti–mouse IgG, and Alexa Fluor 633 goat anti–rat IgG were obtained from Molecular Probes.
Alexa fluor 633 goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
Alexa Fluor 633 goat anti-rabbit IgG is a fluorescently labeled secondary antibody. It binds to rabbit primary antibodies and can be used for detection and visualization in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (4 mentions)
Triton x 100, by Merck Group (3 mentions)
Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Neg 50, by Thermo Fisher Scientific (2 mentions)
Prolong diamond antifade containing dapi, by Thermo Fisher Scientific (2 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Control igg, by R&D Systems (1 mentions)
Alexa fluor 633 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Apc conjugation kit, by Abcam (1 mentions)
Alexa fluor 647 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti rat igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Anti sox9, by Merck Group (1 mentions)
Anti osx, by Abcam (1 mentions)
25 protocols using alexa fluor 633 goat anti rabbit igg
1
Immunophenotyping of CCR8-expressing Cells
2
Multiparametric Flow Cytometry of Adipose Tissue
3
Immunostaining of Zebrafish Embryos
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Embryos at 4 dpf were fixed in ice-cold Cytoskelfix (Cytoskeleton Inc., USA) for 10 minutes, permeabilized in 1.5% Triton X-100 (Sigma, USA) for 1 hour, then blocked with 5% goat serum overnight. Embryos were incubated with anti-Svila (1:200) and anti-acetylated α-tubulin (6-11B1; Sigma, USA), washed in PBS, then incubated with Alexa Fluor 633 goat anti-rabbit IgG (1:200) and Alexa 546 goat anti-mouse IgG (1:200 Invitrogen, USA). Phalloidin labeling of Gt(macf1a–citrine)ct68a/+ fish was as described previously [27 (link)]. Fish were imaged under a 40 × objective on a confocal microscope (Leica, Germany).
4
Antibody Binding in PACT-Cleared Brain
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To demonstrate whether the temperature rise during PACT clearing influences the binding of antibody and antigen, the immunostaining was performed on 1-mm-thick Thy1-GFP-M brain section referring to the original PACT method31 (link). Before staining, the 1-mm-thick slices were cleared with 8% SDS at 37 °C, 42 °C, and 47 °C, respectively. Then the previously cleared samples were immunostained for parvalbumin and nuclei stained with DAPI. After washed in PBST for 1 day and blocked in PBS/0.1% Triton X-100/6% goat serum for 1 day, the samples were transferred to primary antibody dilutions (anti-parvalbumin antibody, Abcam, ab11427, 1:400) for 1 day followed by washing with PBST for several times, then to secondary antibody dilutions (Alexa Fluor 647 goat anti-rabbit IgG, Jackson Immunoresearch, 111-607-003, 1:400) for 1 day at 37 °C. Then the samples were nuclei stained with DAPI at room temperature for 12 hours. The samples were finally washed in PBST for several times before further incubating in 70% (wt/vol) sorbitol solution for 1 or 5 hours. For GFP immunostaining, the cleared brain slice was incubated in primary antibody dilutions (anti-GFP antibody, Millipore, AB3080, 1:200) for 2 days and secondary antibody dilutions (Alexa Fluor 633 goat anti-rabbit IgG, Invitrogen, A-21070, 1:200) for 2 days.
5
Immunofluorescence Staining of Transfected HEK293T Cells
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HEK293T cells (40–60% confluent) grown on coverslips in a 24-well plate were transfected with plasmid DNA using Lipofectamine 2000. At 48 h after transfection, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. For intracellular analysis, cells were permeabilized with 0.1% Triton X-100 in PBS for 8 min. Thereafter, the cells were washed three times with PBS, blocked with 10% FBS in PBS and then incubated with anti-fBST2 pAb diluted 1:500 or anti-HA mAb diluted 1:1000 in PBS containing 1% FBS for 1.5 h at room temperature. Cells were washed three times with PBS and stained with Alexa Fluor 633 goat anti-rabbit IgG (Invitrogen) or Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) diluted 1:1000 in PBS (1% FBS) along with 1 mg/ml 4,6-diamidino-2-phenylindole (DAPI) for 40 min at room temperature. The cells were then washed three times in PBS and incubated with ER-Tracker (http://www.biomart.cn/genecopoeia/index.htm GeneCopoeia, Rockville, MD, USA) for 10 min at room temperature. For surface protein analysis, the same protocol was followed, except the Triton X-100 step was omitted. Stained cells were finally washed three times with PBS and subjected to detection. Fluorescent images were obtained using a Nikon ECLIPSE Ti system with a 20× objective.
6
Isolation and Characterization of Putative Ovarian Stem Cells
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For each FACS, 12 ovaries were digested, blocked and incubated with 1:10 rabbit DDX4C25 antibody (ab13840; Abcam) adjusted to a concentration of 1 mg mL−1, then washed and incubated with 1:250 Alexa Fluor 633 goat anti-rabbit IgG (A21070; Invitrogen, UK). Putative OSCs were sorted using a BD Biosciences FACSAria I (Beckton Dickinson, UK) cytometer, after gating the negative controls (Supplementary Fig. S2 )58 (link). Data were analysed using the FLOWJO software (FLOWJO LLC, USA).
7
Drosophila Neurite and Glia Imaging
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To analyze neurite outgrowth and glial wrapping, we expressed mGFP using a pan-glial driver, repo-Gal4 or repo-LexA, to label glia. The ddaE neurons were labeled by either staining for a neuronal marker, HRP, or by Gal4IG1-1>RFP. Wandering larvae at late 3rd instar stage were dissected and immunostained according to standard protocols. Adult brain dissection and immunostaining was performed following the FlyLight protocols (Janelia Research Campus, HHMI). Larval preparations and adult brains were mounted using anti-fading mounting media (VECTASHIELD®, Vector Laboratories).
Mouse monoclonal antibodies against Nrg180 (BP104, 1:200) were purchased from Developmental Studies Hybridoma Bank (DSHB, University of Iowa). Dylight 405 conjugated anti-HRP (123-475-021, 1:500) was obtained from Jackson Immunoresearch Laboratories. Rabbit anti-Ank2XL was gifted by Dr. Hermann Aberle (Max-Plank Institute for Developmental Biology).59 (link) Guinea pig anti-Pk was gifted by Dr. Jeffrey D. Axelrod (Stanford University).57 (link) Secondary antibodies were Alexa Fluor® 555 Goat anti-mouse IgG (Invitrogen A21422, 1:500), Alexa Fluor® 633 Goat anti-rabbit IgG (Invitrogen A21070, 1:500), Alexa Fluor® 555 Goat anti-Guinea pig IgG (Invitrogen A21435, 1:500). Nuclei were stained with DAPI (Sigma-Aldrich, 28718-90-3).
Mouse monoclonal antibodies against Nrg180 (BP104, 1:200) were purchased from Developmental Studies Hybridoma Bank (DSHB, University of Iowa). Dylight 405 conjugated anti-HRP (123-475-021, 1:500) was obtained from Jackson Immunoresearch Laboratories. Rabbit anti-Ank2XL was gifted by Dr. Hermann Aberle (Max-Plank Institute for Developmental Biology).59 (link) Guinea pig anti-Pk was gifted by Dr. Jeffrey D. Axelrod (Stanford University).57 (link) Secondary antibodies were Alexa Fluor® 555 Goat anti-mouse IgG (Invitrogen A21422, 1:500), Alexa Fluor® 633 Goat anti-rabbit IgG (Invitrogen A21070, 1:500), Alexa Fluor® 555 Goat anti-Guinea pig IgG (Invitrogen A21435, 1:500). Nuclei were stained with DAPI (Sigma-Aldrich, 28718-90-3).
8
Visualizing Zebrafish Hair Cell Dynamics
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Zebrafish at 6 or 14 dpf were incubated with 5 μM FM1-43FX (Invitrogen), a fixable analog of FM1-43, for 30 s, then washed three times at 5 min intervals with the rinsing solution before overnight fixation with 4% paraformaldehyde (PFA) at 4 °C. More specifically, for FM1-43FX uptake, the fish were put into baskets and bathed with labeling solution with manual agitation that resulted in fish movements forward and backward, up and down, and side to side, randomly for 30 s. Permeabilization was performed with 3% Triton-X100 (Sigma) overnight at room temperature. Blocking with 5% goat serum was carried out for 6 h at room temperature. Following this, samples were rinsed with phosphate-buffered saline (PBS) four times for 5 min. To visualize hair cells, parvalbumin 3 antiserum at 1:600 dilution was used as a primary antibody and Alexa Fluor 633 goat-anti-rabbit IgG (Invitrogen) at 1:200 dilution was used as the secondary antibody. To identify hair-bundle orientation, Alexa Fluor 633 phalloidin at 1:50 dilution was used to label actin filaments. Processed zebrafish samples were mounted and kept in antifade mounting medium (VECTASHIELD; Vector Laboratories). Images were acquired on a confocal microscope with excitation wavelengths of 488 nm for FM1-43FX and 633 nm for Alexa Fluor 633.
9
Colonic Mucin Immunodetection on Intestinal Sections
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Sections on microscope slides were treated with a blocking solution (2% goat serum; 1% bovine serum albumin; 0.2% Triton X-100; 0.05% Tween 20) for 1 hour at room temperature. Sections were then incubated with 1:50 dilution of an anti-mouse colonic mucin primary antibody (anti-MCM, a gift of Dr. Ingrid B. Renes, Erasmus MC-Josephine Nefkens Institute) diluted in the blocking solution for 12 hours at 4°C. After incubation, intestinal sections were rinsed for 3 minutes each in fresh 1X PBS, treated with blocking solution for 1 hour at room temperature, and then incubated with a 1:1000 dilution of the Alexa Fluor 633 goat anti-rabbit IgG (Cat# A21070, Invitrogen, Carlsbad, CA) in blocking solution and rinsed for 3 minutes each in fresh 1X PBS.
10
Coculture of HBVP and HUVEC under flow
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HBVP cells were harvested and seeded at a concentration of 1.3 × 105 cells in 0.4 optical plastic flow microslides (80176, Ibidi) precoated with 1 mg/mL gelatin and incubated for 24 hours under standard culture conditions. After the initial incubation, 100 μg/mL of collagen I (354249, Corning) diluted in DMEM cell culture media was added to the slides to create a thin layer on top of the HBVP cells. After 2 hours, the media was removed and 2.5 × 105 HUVECs were seeded on top of the collagen I layer and incubated for additional 24 hours. After cell co-cultures were established, the slides were exposed to laminar (15 dyn/cm2) or pulsatile (12–15 dyn/cm2) flow for 24 hours, implementing a peristaltic pump adapted to produce different types of flow (Abello, Raghavan, Yien, & Stratman, 2022 (link)). After 24 hours, cultures were rinsed with 1x PBS and fixed for 30 minutes in 4% paraformaldehyde at room temperature. Cell cultures were immunostained with α-Smooth muscle actin D4K9N XP® rabbit monoclonal antibody (19245, Cell Signaling), followed by Alexa Fluor 633 goat anti-rabbit IgG (A21071, Invitrogen). Confocal images were obtained using a 40x objective with a W1 Spinning Disk confocal microscope, a Fusion camera, and the Nikon Eclipse Ti2-E base. Fiji image processing software was used for image analysis and fluorescence intensity quantification.
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