For immunofluorescence microscopy of intact cauda epididymal sperm, prepared as described above, sperm were fixed at 4°C in 4% formaldehyde in 0.1 M sodium phosphate buffer, with pH 7.6, for at least 30 minutes, attached to poly-L-Lysine coated coverslips, washed with PBS, and permeabilized by incubation for 10 minutes in −20°C acetone. After three rinses in PBS, nonspecific protein binding sites were blocked in PBS containing 0.1% Tween-20 and 2.5% BSA (blocking solution). Coverslips were then incubated with anti-TEX101 antibody in blocking solution, washed, and incubated with FITC-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in blocking solution. Coverslips were washed with PBS and the cells were examined by phase contrast and epifluorescence microscopy.
Fitc conjugated donkey anti goat igg
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The FITC-conjugated donkey anti-goat IgG is a secondary antibody that binds to goat primary antibodies. It is labeled with the fluorescent dye FITC, allowing for the detection and visualization of goat antibodies in various immunoassays and imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Axiophot, by Zeiss (1 mentions)
24 well microplates, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Fitc conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Anti cd68, by Santa Cruz Biotechnology (1 mentions)
Arginase 1, by BD (1 mentions)
Lsm 700 system, by Zeiss (1 mentions)
Pe conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Bx53 fluorescence microscope, by Olympus (1 mentions)
Microscope cover glass, by NEST Biotechnology (1 mentions)
Texas red conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Beyotime (1 mentions)
8 protocols using fitc conjugated donkey anti goat igg
1
Immunofluorescence Microscopy of Cauda Epididymal Sperm
2
Immunofluorescence Microscopy of Cauda Epididymal Sperm
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For immunofluorescence microscopy of intact cauda epididymal sperm, prepared as described above, sperm were fixed at 4°C in 4% formaldehyde in 0.1 M sodium phosphate buffer, with pH 7.6, for at least 30 minutes, attached to poly-L-Lysine coated coverslips, washed with PBS, and permeabilized by incubation for 10 minutes in −20°C acetone. After three rinses in PBS, nonspecific protein binding sites were blocked in PBS containing 0.1% Tween-20 and 2.5% BSA (blocking solution). Coverslips were then incubated with anti-TEX101 antibody in blocking solution, washed, and incubated with FITC-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in blocking solution. Coverslips were washed with PBS and the cells were examined by phase contrast and epifluorescence microscopy.
3
Immunocytochemical Analysis of hRPE Cells
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hRPE cells in 9th passage were cultured on pre-coated coverslips in a 24-well microplates (Nunc, Roskilde, Denmark) at a density of 1 × 105 cells/well and incubated for 24 h. Cultured cells were fixed using ice-cold methanol (−10°C) (Merck, Darmstadt, Germany) for 5 min at room temperature and then slides were blocked using 1% bovine serum albumin (BSA) (Merck, Darmstadt, Germany) in 1% PBST (1% Triton X-100 in PBS) (Sigma-Aldrich, Munich, Germany) for 20 min at room temperature. Coverslips were incubated in diluted solution of antibodies against Oct4, Chx10, Pax6, and Ki67 (Santa Cruz, Carlsbad, CA, USA) in 1.5% BSA and 1% PBST overnight at room temperature. Then the coverslips were incubated with the secondary antibody in 1.5% BSA and 1% PBST for 1 hour at room temperature in the dark. The secondary antibodies were fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:100, Santa Cruz, Carlsbad, CA, USA), FITC-conjugated goat anti-mouse IgG (1:100, Santa Cruz, Carlsbad, CA, USA), and FITC-conjugated donkey anti-goat IgG. The nuclei of cells were stained using 4’,6-diamidino-2-phenylindole (DAPI) (1 mg/ml, Santa Cruz, Carlsbad, CA, USA) for 1 min. Coverslips were mounted with a drop of mounting medium (90% glycerol, 10% PBS, and 10% (w/v) phenylenediamine). Samples were observed under fluorescence microscope (Axiophot, Zeiss, Germany).
4
Immunofluorescent Staining of Cytoskeletal Markers
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Cells were grown on glass coverslips in 6-well plates for 48 h, then fixed with 3% paraformaldehyde and permeabilised with 0.2% Triton X-100. Immunofluorescent staining was performed according to standard protocols. Treated cells were first probed with anti-HK1, anti-HK2, anti-E-cadherin or anti-vimentin antibody as described above, and then incubated with Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L) (Invitrogen) or FITC conjugated donkey anti-goat IgG (Santa Cruz Biotechnology) secondary antibody, as well as counterstained nuclei with DAPI. For stress fibre F-actin staining, prepared cells were directly stained with Alexa Fluor 488-conjugated phalloidin solution (Molecular Probes, Eugene, OR, USA). Stained cells were examined and imaged using an inverted fluorescence microscope (Olympus IX71; Olympus Co., Tokyo, Japan).
5
Quantitative Analysis of M1 and M2 Macrophages
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Vessel tissue sections were micro cut at room temperature and then deparaffinized through the dewatering process. Subsequently, the sections were triple immunostained with anti-CD68 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), inducible nitric oxide synthase (iNOS, Santa Cruz Biotechnologies) for M1 macrophages, and arginase-1 (BD Biosciences, Franklin Lakes, NJ, USA) for M2 macrophages at 4°C overnight. The sections were washed for 10 min in 1% phosphate-buffered saline (PBS), and then secondarily labeled with FITC-conjugated donkey anti-goat IgG, PE-conjugated goat anti-rabbit IgG, and TR-conjugated goat anti-mouse IgG (Santa Cruz Biotechnologies), respectively, for 1 hour in the dark at room temperature. The sections were washed in PBS for 10 minutes, mounted with Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI, ImmunoBioscience, Mukilteo, WA, USA), and stored in the dark at 4°C until confocal microscopy was performed with an LSM 700 system (Carl Zeiss, Germany). The quantification of CD68, iNOS and Arg1 were analyzed ZEN software. Then we calculated the co-stained area of each iNOS, Arg-1 in CD68 expression within three representative region of interest rectangles per staining slide (n = 4 vessels/group).
6
Immunofluorescence Staining of PAC1 and Thy-1
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The cultured cells were fixed in 4% paraformaldehyde for 30 min. Following 3 washes with PBS, cells were permeabilized in 0.2% Triton X-100 for 30 min, blocked for 1 h in 10% FBS and 1% BSA in PBS, and then incubated overnight at 4°C with primary antibodies against PAC1 (1∶200, sc-15964, Santa Cruz, USA) and Thy-1 (1∶200, sc-19614, Santa Cruz, USA). For control experiments, primary antibodies were omitted. The next day, cells were washed 3 times with PBS and incubated with secondary FITC-conjugated donkey anti-goat IgG or DyLight 594-conjugated goat anti-mouse IgG (1∶200, Santa Cruz, USA) for 1 h, and then counterstained with DAPI in 1% BSA in PBS at room temperature. Images were obtained with a fluorescence Olympus BX51 microscope (Olympus, Tokyo, Japan).
7
Apoptosis Imaging of NDV-Infected Cells
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The BHK-21 cells at a concentration of 3 × 10 5 and infected with an NDV MOI of 1. The cells were challenged with NDV at 0 and 24 h, the caspase and caspase inhibitor groups were incubated separately. The sample was added to Mitotracker (Invitrogen, M-7512) (50 μg/mL) and incubated at 37°C for 30 min, after which the medium was removed and the cover slip was washed with cold PBST. After that, the cells were fixed with 3.7% ice-cold paraformaldehyde for 10 min. Following, the slip washed three times with cold PBST. The sample was added to the primary antibodies (goat anti-AIF and goat anti-Endo G Santa Cruz, USA; chicken anti-ND Abcam ○ R , UK) in PBST for at least 4 h at room temperature. After washing, the cells were incubated with FITC-conjugated donkey anti-goat IgG (Santa Cruz, USA) or goat anti-chicken IgY (Santa Cruz, USA) in PBST at room temperature for 1 h in the dark. Then, the cells were added to DAPI 1 µg/ µL in PBST at room temperature for 25 min in the dark. Finally, the cells were placed under the cover slip sealed with 40% glycerol, and visualization was carried out using a fluorescence microscope.
8
Immunofluorescent Labeling of Myogenic Markers
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For immunofluorescent labeling, cells were firstly plated on microscope cover glass (Nest, China), and then fixed in cold acetone for 10 min, permeabilized with 0.1% Triton X-100 for 10 min before washed twice in PBS and incubated with Rabbit polyclonal MyoD (1:200, Santa Cruz); Rabbit polyclonal myogenin (1:200, Santa Cruz); Rabbit polyclonal anti-p-CaMKIV (phosphor T200, 1:200, Abcam, USA); Rabbit polyclonal anti-CaMKIV (1:200, Abcam, USA); Mouse anti-mouse H-2K b (1:200, BD Biosciences); Mouse monoclonal H2-Ea (1:200, Santa Cruz); Goat polyclonal TLR3 (1:200, Santa Cruz), respectively. FITC-conjugated donkey antigoat IgG (1:400, Santa Cruz), Texas Red-conjugated goat anti-rabbit IgG (1:400, Santa Cruz), Rhodamineconjugated rabbit anti-mouse IgG (1:400, Santa Cruz) , or Alexa Fluor 488 goat anti-rabbit IgG (1:500, Beyotime, China) were used as secondary antibodies. Nuclei were counterstained with DAPI (Santa Cruz). Slides were then viewed under Olympus BX53 fluorescence microscope (Olympus, Japan).
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