Processing of transfected cells for confocal microscopy was performed as we described previously. Briefly, monolayers were permeabilized and fixed using 100% methanol held at –20°C for 5 min and were then blocked and labeled with mouse-anti-FLAG (Sigma) (1/200 dilution in 3% bovine serum albumin [BSA]–phosphate-buffered saline [PBS]) and rabbit-anti-Myc (Proteintech) (1/200 dilution in 3% BSA–PBS). Cells were counterlabeled with Alexa Fluor 488 anti-mouse antibody (Invitrogen) (1/4,000 dilution in 3% BSA–PBS), Alexa-Fluor 555 anti-rabbit antibody (Invitrogen) (1/4,000 dilution), and DAPI (4′,6-diamidino-2-phenylindole) to stain the nuclei. Monolayers were examined by confocal microscopy. A total of 100 cells for each replicate were counted to determine the presence or absence of localization.
Alexa fluor 555 anti rabbit antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 555 anti-rabbit antibody is a fluorescently labeled secondary antibody used for detecting rabbit primary antibodies in various immunoassays and imaging applications. The antibody is conjugated with the Alexa Fluor 555 dye, which exhibits bright red fluorescence.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 secondary anti rat, by Thermo Fisher Scientific (2 mentions)
Rabbit anti myc, by Proteintech (1 mentions)
Alexa fluor 488 anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti flag, by Merck Group (1 mentions)
Nie microscope, by Yokogawa (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Prime 95b camera, by Teledyne (1 mentions)
Nis element, by Nikon (1 mentions)
Anti p erk, by Merck Group (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
D1d3g, by Cell Signaling Technology (1 mentions)
Cellmask deep red plasma membrane stain, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Ab16048, by Abcam (1 mentions)
Ab30637, by Abcam (1 mentions)
Ab50595, by Abcam (1 mentions)
Mowiol, by Merck Group (1 mentions)
8 protocols using alexa fluor 555 anti rabbit antibody
1
Confocal Microscopy of Transfected Cells
2
Quantifying DNA Damage in Zebrafish Embryos
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Fixed zebrafish embryos were permeabilized with acetone for 1 h at − 20 °C, blocked with 10% NDS in 0.4% PBST for 1 h at 37 °C and immunostained with primary rabbit anti-γH2AX (1/500 in 0.4% PBST, GeneTex (Irvine, US), Supplementary table 1, online resource) for 3 h at 37 °C. Embryos were incubated overnight at 4 °C with secondary Alexa Fluor 555 anti-rabbit antibody (1/1000 in 0.4% PBST, Invitrogen). In-between washing steps were performed with 0.4% PBST. Nuclei were stained using NucBlue counterstaining (Invitrogen) and embryos were mounted using ProLong Gold antifade reagent (Invitrogen). For image acquisition, a Nikon NiE microscope was used with a 40x objective, equipped with a Yokogawa CSU-X spinning-disk module and Teledyne Photometrics Prime 95B camera. The setup was controlled by NIS-Elements (software version 5.30.05, Nikon Instruments Europe B.V.). For image analysis, the number of neuronal nuclei positive for γH2AX and located in the spinal cord of zebrafish embryos was counted and normalized to the length of the spinal cord section using ImageJ.
3
Zebrafish Tbx6 Protein Immunostaining
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For immunostaining of zebrafish Tbx6 proteins, we generated anti-rabbit antibody against zebrafish Tbx6. The immunogen was prepared from E. coli. expressing a fragment of the zebrafish Tbx6, ranging from the 561st to the 874th position in its amino-acid sequence. Purified proteins electroeluted from poly-acrylamide gel were used to immunize 2 rabbits. After 7 injections of the purified proteins, sera (#1 and #2) were recovered from the rabbits; and their reactivity and specificity were assessed by Western blotting (Figure S1 ). Whole mount immunostaining was conducted using one of the antisera (#1) at a dilution of 1∶200 in 2%BSA-PBS containing 0.1% Triton-x100, with incubation for 48 hrs at 4°C and detected with alexa fluor-555 anti-rabbit antibody (Invitrogen). For quantification of the expression levels of tbx6 mRNA and Tbx6 protein in the PSM, signal intensity was measured by ImageJ software (National Institute of Health) and background was subtracted. Obtained intensity values were normalized to a range between 0 and 1. Immunostatining with anti-pErk (Sigma) was performed according to the protocol by [32] (link). To compare Tbx6 protein pattern with tbx6, her1, mesp-ab, mesp-ba, ripply1, and ripply2 mRNAs, immunostaining was performed after in situ hybridization.
4
Visualizing Nuclear Envelope and cGAS Localization
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Cells were fixed in 4% paraformaldehyde (Sigma) for 15 min at room temperature. Cells were then permeabilized in 0.25% (vol/vol) Triton-X for 10 min at room temperature and blocked in staining buffer, 5% FBS, 2% BSA (HyClone), and 0.1% (vol/vol) Triton-X (Merck) in 1× PBS for 30 min at room temperature. For visualization of nuclear envelope, cells were stained with rabbit anti-lamin B1 antibody (ab16048, Abcam) overnight at 4 °C and was subsequently stained with secondary antibody Alexa Fluor 488 anti-rabbit antibody (Invitrogen) at room temperature for 2 h. For cGAS localization to micronuclei, cells were stained with rabbit anti-cGAS antibody (D1D3G, Cell Signaling Technologies) overnight at 4 °C and followed by Alexa Fluor 555 anti-rabbit antibody (Invitrogen) at room temperature for 2 h. Cells were then stained with 10 μg/mL Hoechst 33342 (ThermoScientific) and 1:10,000 diluted CellMask Deep Red Plasma membrane stain (Invitrogen). In between each incubation step, the cells were washed with 0.1% Triton-X (Merck). The coverslips were mounted onto clean glass slides with ProLong Gold Antifade (Invitrogen) and imaged with Zeiss LSM710 Confocal microscope using a 63× objective lens.
5
Visualizing Golgi Apparatus in Drosophila Larvae
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CRISPR-mutations third-instar larvae raised at 25°C were harvested, dissected in PBS 1×, fixed in 4% paraformaldehyde for 15 min and washed twice in PBS containing 0.3% Triton X-100 (Sigma-Aldrich). Dissected larvae were probed with anti-GM130 (Abcam, ab30637, rabbit, 1:1000) overnight at 4°C, then washed 3 times with PBS plus 0.3% Triton X-100 and incubated with Alexa Fluor 555 anti-rabbit antibody (Molecular Probes Invitrogen, 1:500). Larvae were washed 3 times with PBS and mounted on coverslips using Mowiol (Sigma-Aldrich).
HeLa cells transfected as indicated above were fixed in PBS containing 4% paraformaldehyde for 15 min, incubated with 50 mM NH4Cl for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 3 min and then blocked with 2% BSA and 0.2% gelatin for 30 min. Antibodies used were anti-GM130 (BD, #610822, mouse, 1:1000); anti-TGN46 (Abcam, ab50595, rabbit, 1:200). Alexa Fluor 555 conjugated goat anti-mouse and Alexa Fluor 647 conjugated goat anti-rabbit (Molecular Probes Invitrogen) were applied for 1 h at room temperature. Coverslips were mounted using Mowiol (Sigma-Aldrich).
All confocal images were acquired using a Leica TCS SP5 II confocal microscope, equipped with a PlanApo 100×/1.4 Oil objective, using a 543 nm laser line. Confocal microscopy imaging was performed at 1024 × 1024 pixels per image, with a 200 Hz acquisition rate.
HeLa cells transfected as indicated above were fixed in PBS containing 4% paraformaldehyde for 15 min, incubated with 50 mM NH4Cl for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 3 min and then blocked with 2% BSA and 0.2% gelatin for 30 min. Antibodies used were anti-GM130 (BD, #610822, mouse, 1:1000); anti-TGN46 (Abcam, ab50595, rabbit, 1:200). Alexa Fluor 555 conjugated goat anti-mouse and Alexa Fluor 647 conjugated goat anti-rabbit (Molecular Probes Invitrogen) were applied for 1 h at room temperature. Coverslips were mounted using Mowiol (Sigma-Aldrich).
All confocal images were acquired using a Leica TCS SP5 II confocal microscope, equipped with a PlanApo 100×/1.4 Oil objective, using a 543 nm laser line. Confocal microscopy imaging was performed at 1024 × 1024 pixels per image, with a 200 Hz acquisition rate.
6
Quantitative Analysis of γH2AX Foci
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Cells were grown on coverslips in 12-well plates. After irradiation, cells were fixed with 4% formaldehyde in PBS at room temperature for 10 min and methanol at -20°C for 20 min, permeabilized in 0.1% Triton X-100 in PBS for 10 minutes, and blocked with 5% skim milk for 1 h. Cells were then incubated at room temperature for 2 hours with anti-γH2AX (Cell Signaling Technology, Beverly, MA, USA). Cells were then stained with Alexa Fluor® 555 anti-rabbit antibody (Molecular Probes, Eugene, OR, USA) at 37°C for 1 hour. Following extensive washing in PBS, the cells were mounted on slides using DAPI mounting medium (Eugene, OR, USA). The stained cells were observed under an Olympus IX71 microscope (Olympus, Tokyo, Japan) in NIRS while under a laser scanning confocal microscope (Olympus FV1200, Tokyo, Japan) in Soochow University. At least 100 cells were scored for each sample.
7
Analyzing Gut Microbiome in Colon Adenomas
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To examine the mucus barrier, tissues were fixed in Carnoy’s solution (Ricca Chemical, Arlington, TX, USA). Analysis of bacteria in the lamina propria of adenomas or normal colonic mucosa was performed in PFA-fixed tissue sections. Paraffin-embedded slides were de-paraffinized and hybridized to universal eubacterial probe (5’-GCTGCCTCCCGTAGGAGT-3’) or a control probe (5’-CGACGGAGGGCATCCTCA-3’) labeled with Alexa 488. All probes were obtained from IDT Technologies (Coralville, IA, USA). Hybridization was performed overnight at 56C, followed by washing and counterstaining with the nuclear dye Hoechst 33342. For concurrent FISH and immunofluorescence staining, slides were stained for eubacteria FISH probe, as described earlier. The antigen retrieval step was excluded. COX-2 and F4/80 staining was performed as follows. After a 30-min blocking step with BSA, COX-2 and F4/80 antibodies were diluted, applied to the samples, and incubated in a humidifier chamber overnight at 4°C. Alexa Fluor 647 secondary anti-rat (Thermo Fisher Scientific) or Alexa Fluor 555 anti-rabbit antibodies (Thermo Fisher Scientific) were added and incubated at room temperature for 30 min. After 3 washes in PBS and the addition of Hoechst 33342 nuclear stain, coverslips were mounted on slides.
8
Analyzing Gut Microbiome in Colon Adenomas
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