Antibodies, used for immunostaining: rabbit anti-SYCP3 antibodies (diluted 1:250, Abcam, Cambridge, UK) as a marker for lateral elements of SC and axial elements; human anti-centromere antibody CREST (1:250, Fitzgerald Industries International, Concord, MA, USA) for detecting kinetochores. Localization of CDK2 was detected applying mouse antibodies CDK2 (1:250, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). DNA double-strand break (DSB) loci were immunostained with mouse antibodies against the RAD51 protein (1:200, Abcam, Cambridge, UK). Late recombination nodules were detected using mouse antibodies MLH1 (1:50, Abcam, Cambridge, UK). Proteins involved in the telomere attachment to nuclear envelope SUN1 were distinguished using rabbit antibodies SUN1 (1:250, Abcam, Cambridge, UK). As secondary antibodies we used goat anti-rabbit IgG, Alexa Fluor 488-conjugate (Invitrogen, Carlsbad, CA, USA); goat anti-human IgG, Alexa Fluor 546-conjugate (Invitrogen, Carlsbad, CA, USA); goat anti-mouse IgG, Alexa Fluor 546-conjugate and IgG, Alexa Fluor 555-conjugate (Invitrogen, Carlsbad, CA, USA) (diluted 1:300–800). Slides were washed in phosphate-buffered saline (PBS) and immersed into Vectashield with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Slides were analyzed using a fluorescence light microscope Axio Imager D1 (Carl Zeiss, Jena, Germany).
Alexa fluor 546 conjugate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 546-conjugate is a fluorescent dye used in various applications, including cell imaging, flow cytometry, and immunoassays. It has an excitation maximum at 556 nm and an emission maximum at 573 nm, making it suitable for detection in the orange-red region of the visible spectrum.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Im 1000, by Leica (2 mentions)
4 well chamber slide, by BD (2 mentions)
Confocal microscope tcs sp 8 x, by Leica (1 mentions)
Application suite, by Leica (1 mentions)
Prolong gold antifade containing dapi, by Thermo Fisher Scientific (1 mentions)
Texas red x phalloidin, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Biotinylated anti brdu antibody, by BD (1 mentions)
Alexa fluor 488 goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Axio imager d1, by Zeiss (1 mentions)
Alexa fluor 555 conjugate, by Thermo Fisher Scientific (1 mentions)
Vectashield with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
P c jun, by Santa Cruz Biotechnology (1 mentions)
C jun, by Cell Signaling Technology (1 mentions)
Mouse anti β tubulin antibody, by Merck Group (1 mentions)
10 protocols using alexa fluor 546 conjugate
1
Immunostaining Assay for Meiotic Protein Localization
2
Visualizing NFAT1 Localization in TZM-bl Cells
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TZM-bl cells were cultured onto 15 mm cover slips (MENZEL-GLÄSER Lot# 94711285, Germany) and treated with 240μM of INDY (Glixx) or the DMSO control. After twenty-four hours of incubation, cells were washed with PBS and fixated with 70% ice cold ethanol for 10 min. After fixation, the cells were washed with PBS and incubated with 5μg of anti-NFATc1 antibody (H-110: Santa Cruz, USA) for 30 min at 4°C. Next, cells were blocked for 30 min with PBS containing 0.5% bovine serum albumin. After a wash with PBS, cells were incubated with the secondary antibody: 1: 400 Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 546 conjugate (#A10040, Invitrogen) and Hoechst 1:10,000 (H1398, Invitrogen) for 45 min at room temperature. Images were captured using a Leica confocal microscope TCS SP-8 X (Leica Microsystems, USA) and analyzed and processed using Leica Application Suite (Leica Microsystems).
3
Immunofluorescence Staining of MAML3 and β-Catenin
4
BrdU-labeling of Pancreatic Islet Cells
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Isolated islets were incubated with 10 µM BrdU during the last 24 h according to the manufacturer’s protocol. At the completion of incubation, the islets were collected in a collodion bag consisting of the inner wall surfaces of tubes, fixed with 10 % formalin, and embedded in paraffin60 (link). Islet sections were made at an interval of 20 µm and the labeled cells were immunostained with biotinylated anti-BrdU antibody (51–75512 L, BD Bioscience, San Jose, CA, USA) according to the BrdU In Situ Detection Kit protocol (BD Bioscience). Immunoreactivity was visualized by incubation with a substrate solution containing 3,3′-DAB. For analysis, approximately 3000–8000 nuclei of islets were counted. For fluorescent immunostaining, monoclonal anti-insulin antibody (I2018, Sigma) and biotinylated anti-BrdU antibody (51–75512 L, BD Bioscience) were used as primary antibodies, and Alexa Fluor 488 goat anti-mouse IgG1 (A21121, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 546 conjugate (S11225, Invitrogen) were used as secondary antibodies.
5
Immunohistochemical Analysis of Sigma-1 Receptor
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Enucleated eyes were fixed and sectioned. Antigen retrieval was used and involved the use of 6 M urea in 0.1 M Tris pH 9.5 at 80°C for 10 minutes. Tissue sections were blocked with 5% normal donkey serum and 5% BSA + 0.1% Triton X-100 for 1 hour at room temperature and incubated with appropriate primary antibodies: affinity-purified σ-1r monoclonal antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or polyclonal antibody (1:100 dilution; Santa Cruz Biotechnology); RBPMS polyclonal antibody (1:100 dilution; GeneTex, Inc. Irvine, CA, USA), c-Jun (1:200, Cell Signaling #9165s), p-c-Jun (1:50, Santa Cruz sc-822), Caspase-3 (1:500, Cell Signaling #9664) at 4°C overnight. Coverslips were then washed three times with PBS, and a 1:1000 dilution of secondary antibodies donkey anti-rabbit or anti-mouse IgG (Alexa Fluor 647 or 488) conjugate and donkey anti-mouse IgG or anti-rabbit IgG (Alexa Fluor 546) conjugate (Invitrogen, Carlsbad, CA, USA) were added and incubated for 1 hour in the dark at room temperature. After incubation, the coverslips were washed three times with PBS. Mounting was performed on glass slides using antifade reagent with DAPI (P36931 Prolong Gold; Invitrogen) and allowed to dry for overnight in the dark. Tissues were imaged using a (LSM 510; Zeiss, Thornwood, NY, USA) at 40 times magnification.
6
Investigating Retinoic Acid and Arp2/3 Regulation
7
Immunofluorescence Staining of Cytoskeleton Proteins
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0.5-1×104 cells were plated into 24-well plates containing glass cover slips coated with 0.2% gelatin. After 6–24 h, culture medium was removed, ice-cold methanol added to each well and plates incubated for 20 min at 4 °C. Cells were washed twice with PBS, and incubated in 0.2% Triton for 20 min with rotation. Cells were blocked in 2% FBS in PBS for 1 h. Primary antibodies for Tpm1.6/7 (1:200, from P.W.G.), Tpm1.8/9 (1:200, from P.W.G.), Arp2 (1:200, #ab47654, Abcam) were added and incubated O/N at 4 °C. Cells were washed twice with PBS, and secondary antibodies [goat anti-rat Alexa Fluor 488 conjugate (1:250, #A10528, Life Technologies); goat anti-rabbit Alexa Fluor 546 conjugate (1:250, #A11035, Life Technologies); goat anti-mouse Alexa Fluor 647 conjugate (1:250, #A32728, Life Technologies)] and DAPI (#D1306, Thermo Fisher Scientific) added. Slides were mounted using VECTASHIELD® (#H100010, VECTOR laboratories) and cells imaged using a LSM-700 (Zeiss) with 20 , 40 , 63 lenses. Images were analyzed using ImageJ.
8
Immunofluorescence Assay for FGF8 Expression
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RH30 and RD cells were seeded on 4-well chamber slide (Falcon) and incubated at 37°C for 48 hours. Once reached sub-confluence, cells were fixed with 4% paraformaldehyde for 20 minutes, and permeabilized with 0,2%Triton X-100 in PBS1X for 10 minutes. Slides were then incubated for 10 minutes in 100 mM glycine and for further 10 minutes in 5% BSA in PBS1X. Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes. Slides were washed and mounted in 1:1 glycerol/PBS1X supplemented with DAPI 1:500 (6,6-diamino-2-phenylindole, dihydrochloride) (Thermo Fisher scientific). The images were acquired with a Leica DFC420 digital camera, mounted on a Leica DM4000B microscope, at 20X magnification. Image analysis was performed with Leica IM1000 software (Leica Microsystem).
9
Immunofluorescence Staining of GTF2i
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RH30 and RH4 cells were seeded on 4-well chamber slide (Falcon) and incubated at 37°C for 48 h. Once reached sub-confluence, cells were fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.2%Triton X-100 in PBS1X for 10 min. Slides were then incubated for 10 min in 100 mM glycine and for further 10 min in 10% FBS in PBS1X. Primary antibody against GTF2i (Thermo Fisher scientific) and in 2%FBS/1X PBS were probed at 37°C for 60 mins, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in 1X PBS at 37°C for 60 min. Slides were washed and mounted in 1:1 glycerol/1X PBS supplemented with DAPI (6,6-diamino-2-phenylindole, dihydrochloride) (Thermo Fisher scientific). The images were acquired with a Leica DFC420C digital camera, mounted on a Leica DM4000B microscope, at 20X magnification. Image analysis was performed with Leica IM1000 software (Leica Microsystem).
10
Quantifying Glucose Transporter Expression
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B16-F10 and Mel16 melanoma cells were fixed either with 4% paraformaldehyde followed by 0.1% Triton-X-100 to allow permeabilization or with cold methanol at −20 °C. Cells were then incubated with the primary antibody anti-glucose transporter Glut-1 rabbit monoclonal (1:100) (ab115730, Abcam, Cambridge Science Park, Cambridge, UK). The primary antibody was visualized using a goat anti-rabbit Alexa Fluor 546 conjugate (1:800) (ThermoFisher Scientific, S.r.l. Rodano, Milan, Italy). For immunofluorescence staining of the F-actin, the cells were incubated with TRITC-phalloidin (1:400) (P1951, Sigma-Aldrich). Coverslips were then mounted using ProLong Gold antifade reagent with DAPI (InVitrogen, Life Technologies, Monza, Italy). Samples were examined with an Apotome system (Zeiss, Oberkochen, Germany) connected to an Axio Observer inverted fluorescence microscope (Zeiss). Quantitative analysis of fluorescence signal was performed using the Zen 2.6 (blue edition) software (Zeiss). The results are expressed as mean fluorescence intensity/cell ± SD relative to the untreated sample value, which was set as 1.
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