Mouse anti-caspase-1 mAb (AG-20B-0042), which recognizes both full-length and cleaved p20 fragments, was purchased from Caltag Medsystems (Buckingham, UK). Goat polyclonal anti-cleaved caspase-1 p20 was obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Rabbit anti-cleaved caspase-3 mAb (5A1E), HRP conjugated goat anti-rabbit and anti-mouse IgG were from New England Biolabs (Hitchin, UK). Rabbit polyclonal anti-LC3B, mouse anti-RPE65 mAb (401.8B11.3D9), DyLight® 488 conjugated donkey anti-rabbit IgG and Alexa Fluor 405-donkey anti-goat IgG were from Abcam (Cambridge, UK). Rabbit polyclonal anti-Iba1 was obtained from Wako Chemicals (Neuss, Germany). Alexa Fluor 488 conjugated rabbit anti-goat IgG, Alexa Fluor 488 conjugated goat anti-rabbit IgG and Alexa Fluor 555 conjugated goat anti-mouse IgG were from Life Technologies.
Alexa fluor 488 conjugated goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Japan, Denmark, China
Alexa Fluor 488-conjugated goat anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. It is conjugated to the Alexa Fluor 488 fluorescent dye, which can be detected using fluorescence-based techniques.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (37 mentions)
Alexa fluor 594 conjugated goat anti mouse igg, by Thermo Fisher Scientific (33 mentions)
Alexa fluor 568 conjugated goat anti mouse igg, by Thermo Fisher Scientific (28 mentions)
Lsm 780 confocal microscope, by Zeiss (21 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (19 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (16 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (16 mentions)
Alexa fluor 555 conjugated goat anti mouse igg, by Thermo Fisher Scientific (12 mentions)
Hoechst 33342, by Thermo Fisher Scientific (11 mentions)
Triton x 100, by Merck Group (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (10 mentions)
Lsm 780, by Zeiss (10 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (9 mentions)
Lsm 710, by Zeiss (8 mentions)
Alexa fluor 568 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (7 mentions)
Alexa fluor 546 conjugated goat anti mouse igg, by Thermo Fisher Scientific (7 mentions)
Alexa fluor 546 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (6 mentions)
Alexa fluor 647 conjugated goat anti mouse igg, by Thermo Fisher Scientific (6 mentions)
Paraformaldehyde (pfa), by Merck Group (6 mentions)
Lsm 700, by Zeiss (6 mentions)
389 protocols using alexa fluor 488 conjugated goat anti rabbit igg
1
Detecting Apoptosis Pathway Proteins
2
Immunohistochemical Identification of Macrophage Subsets
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To identify the macrophages by immunohistochemistry, the sections were covered by blocking buffer (5% BSA) for 30 min at room temperature followed by primary and secondary antibody incubation for 1 h each at room temperature. Macrophages were identified by immunofluorescence staining with rat anti-mouse F4/80 monoclonal antibody (CI: A3-1, Bio-Rad) followed by Alexa Fluor® 594 conjugated goat anti-rat IgG (Invitrogen, CA, United States). M1 pro-inflammatory macrophages were further identified by rabbit anti-mouse inducible nitric oxide synthase (iNOS) polyclonal antibody (Abcam, Cambridge, MA, United States) followed by Alexa Fluor® 488 conjugated goat anti-rabbit IgG (Invitrogen, CA, United States). M2 anti-inflammatory macrophages were identified by rabbit anti-mouse liver Arginase (Arg1) polyclonal antibody (Abcam, Cambridge, MA, United States) followed by Alexa Fluor® 488 conjugated goat anti-rabbit IgG (Invitrogen, CA, United States). ProLong Gold Antifade Mount with DAPI (Life Technologies, Grand Island, NY, United States) was used to mount the slides. A fluorescence microscope (Digital Microscope, Keyence, IL, United States) was used to detect the immunohistochemistry staining. Finally, the cells were manually counted in 3 randomly selected fields of view by Image J software (National Institutes of Health, United States).
3
Immunofluorescent Staining of Cadherin Proteins
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Cells in 2D monolayers grown on glass coverslips were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and then incubated with either anti-E-cadherin rabbit monoclonal antibody (1:200; Cell Signaling Technology Inc.) or anti-Pan-cadherin rabbit polyclonal antibodies (1:100; Sigma-Aldrich). The secondary antibody used was AlexaFluor 488-conjugated goat anti-rabbit IgG (ThermoFisher Scientific). Nuclei were stained with DAPI (ThermoFisher Scientific). Images were obtained by confocal microscopy (Leica equipped with a ×40 water immersion objective).
Multicellular spheroids were fixed with 4% paraformaldehyde for 60 min permeabilized with 1% Triton X-100 for 2 days. They were incubated for 24 h with E-cadherin rabbit polyclonal antibodies (1:200; Cell Signaling Technology Inc.) at 4°C. The secondary antibody used was AlexaFluor 488-conjugated goat anti-rabbit IgG (ThermoFisher Scientific). Nuclei were stained with Hoechst 3342 (Invitrogen). Images were obtained by confocal microscopy (Zeiss LSM780 with a ×20 water immersion objective).
Multicellular spheroids were fixed with 4% paraformaldehyde for 60 min permeabilized with 1% Triton X-100 for 2 days. They were incubated for 24 h with E-cadherin rabbit polyclonal antibodies (1:200; Cell Signaling Technology Inc.) at 4°C. The secondary antibody used was AlexaFluor 488-conjugated goat anti-rabbit IgG (ThermoFisher Scientific). Nuclei were stained with Hoechst 3342 (Invitrogen). Images were obtained by confocal microscopy (Zeiss LSM780 with a ×20 water immersion objective).
4
Immunostaining of hESC-Derived Ventricular Cardiomyocytes
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For staining of hESC-VCMs. adherent cells were fixed for 15 min at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS). After washing with PBS, cells were permeabilized in PBS containing 1% Triton X-100 and subsequently blocked in 3% bovine serum albumin. Mitofusin-2 and ROMK1 (KCNJ1) antibodies were purchased from Abcam (Cambridge, MA, USA). Primary antibodies were diluted in PBS with 1% BSA at 1:200 and incubated at RT for 2 hours. Alexa Fluor (AF) 488-conjugated goat anti-rabbit IgG or AF555 anti-mouse IgG (Invitrogen) were used as secondary antibodies and stained for 1 hour at RT. Coverslips were mounted onto glass slides in Prolong Gold mounting medium with DAPI (Invitrogen) and samples were imaged on LSM Carl Zeiss 510 Meta (Carl Zeiss, Jena, Germany). Images were then analyzed and circularity index for each cell given by Image J (NIH, USA).
5
Immunofluorescence Staining of Tissue Samples
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Samples were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4°C. After rinsing with PBS, they were permeabilized in PBS containing 1% Triton X-100 and subsequently blocked in 1% bovine serum albumin (BSA). Primary antibodies (Abcam) were diluted in PBS with 1% BSA at 1:200 and incubated at room temperature for 2 h. Alexa Fluor (AF) 488-conjugated goat anti-rabbit IgG or AF555 anti-mouse IgG (Invitrogen) were used as secondary antibodies at 1:1000 dilution and incubated for 1 h at room temperature. Prolong Gold mounting medium with DAPI (Invitrogen) was used to mount the samples under coverslips. The stained samples were then imaged on LSM Carl Zeiss 700 (Carl Zeiss).
6
Visualizing TC Adhesion and Transmigration
7
Immunofluorescent Analysis of Telomeres
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HEK293T cells were seeded and cultured overnight on 3-aminopropyl triethoxysilane (APTES, Sigma) coated glass slides. The cells were fixed for 20 min in 3.7% formaldehyde (Fisher Scientific), permeabilized using 0.25% Triton X-100 and blocked with 4 × SSC/4%BSA for 1 h at room temperature. Rabbit antibodies against PGP9.5 (UCHL1) (Abcam) and mouse antibodies against TRF2 (Novus Biologicals) were incubated for 1 h and then for 1 h again at room temperature with secondary antibodies Alexa Fluor (AF) 488 conjugated goat anti-rabbit IgG and AF594 conjugated goat anti-mouse IgG, respectively (both Life technologies). Slides were then counterstained with DAPI (Sigma), mounted with Fluoromount G (Southern Biotech) and imaged with a Zeiss Z1 microscope using a 63x oil immersion objective with NA of 1.4 and image J software.
8
Immunofluorescent Analysis of HMGA2 and TRF2
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Cells were seeded and cultured overnight on 3-aminopropyl triethoxysilane (APTES, Sigma) coated glass slides. The cells were fixed for 20 min in 3.7% formaldehyde (Fisher Scientific), permeabilized using 0.25% Triton X-100 and blocked with 4x SSC/4%BSA for 1 h at RT. Primary antibodies for HMGA2 (D1A7) (rabbit monoclonal, Cell Signaling) and TRF2 (mouse monoclonal, Novus Biologicals) were incubated for 1 h at RT followed by 1 h incubation at RT with secondary antibodies such as Alexa Fluor (AF) 488 conjugated goat anti rabbit IgG and AF594 conjugated goat anti mouse IgG (both Life Technologies). Slides were then counterstained with DAPI (Sigma), mounted with Vectashield (Vector Laboratories, Burlington, ON) and imaged with a Zeiss Z1 microscope using a 63x oil immersion objective with NA of 1.4 and Axio Vision Software (Zeiss, Jena, Germany). Following deconvolution, colocalization analysis was performed using the ImageJ colocalization plugin on single, segmented nuclei as described previously [71 (link)]. The colocalizing signals were extracted and displayed as a separate image. 50 nuclei were randomly analysed under each experimental condition and the average number of colocalizing spots per nucleus was graphed with error bars representing standard errors of the means.
9
Immunofluorescence Analysis of Hypoxia-Related Proteins
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Pulp tissues were fixed with 4% paraformaldehyde at 4 °C overnight, after which they were embedded in Tissue-Tek® O.C.T. Compound (Sakura Finetek, Tokyo, Japan). Six-micrometer-thick frozen sections were prepared. Anti-HIF1α rabbit polyclonal antibody (1:500, GTX127309; GeneTex), anti-BCL9 rabbit polyclonal antibody (1:100, PA5-93229; Thermo Fisher Scientific), and anti-β-catenin mouse IgG1κ monoclonal antibody (1:3200, 37447; Cell Signaling Technology) were used for primary antibodies, and samples were incubated with one of the primary antibodies overnight at 4 °C. Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500; Molecular Probes, Eugene, OR, USA) or Alexa Fluor 594-conjugated goat anti-mouse IgG (1:500; Molecular Probes) was used as a secondary antibody and applied for 60 min at room temperature. Mounting Medium with DAPI (Abcam, Cambridge, UK) was used for nuclear staining.
10
Macrophage Phagocytosis Assay with iRBCs
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Adherent differentiated macrophages on round glass coverslips in 24-well plastic plates were incubated with iRBCs for phagocytosis. After a 2-h incubation, the coverslips were washed three times with PBS and fixed with 4% paraformaldehyde solution for 20 min. Cells were permeabilized with 0.3 triton X100 (Sigma) in PBS for 5 min, and then incubated with the primary antibody (anti-DEFB130; Santa Cruz Biotechnology, CA, USA) diluted in 3% fetal calf serum in PBS (1:100) for 1 h at 37 °C in a moist chamber. A secondary antibody of Alexa-Fluor® 488-conjugated goat anti-rabbit IgG (Molecular Probes, Invitrogen, Carlsbad, CA, USA) at a dilution of 1:400 was applied to the coverslips and incubated for 30 min at 37 °C. Hoechst was used to label the nuclear DNA of the cells (Molecular Probes). Coverslips were mounted (Dako, Denmark) and analyzed by use of a confocal laser scanning microscope (TCS NT, Leica, Heidelberg, Germany).
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