For fluorescent immunohistochemistry, tissue samples of embryos and larvae were processed for cryosectioning. Fixed embryos and larvae were incubated in 30% sucrose in PBS overnight at 4°C. The samples were mounted in a mold with Neg-50TM frozen section medium (Fisher Scientific) and sectioned at 20–30μm using a cryostat (Leica CM 1950). Slides were washed twice with PBS for 10min each. The samples were incubated for 1h with blocking solution (5% BSA, 5% heat-inactivated Bovine Serum/HI-BS in PBS). Primary antibodies include anti-pan myosin MF20 (DSHB), Pax7 (DSHB) and pan-neural (anti HNK-1) Zn-12 antibodies at a 1/20 dilution respectively for overnight incubation. The slides were washed three times with blocking solution for 1h each and incubated overnight with Anti-mouse IgG Alexa 488 (1:1000) (Life Technology) and Hoechst (1:2000) (Life Technology). The slides were washed three times with blocking solution for 1h each and with PBSTx twice for 15min each. Mounting reagent (Invitrogen) was placed on tissues and covered with cover slip. Images were captured using a Zeiss LSM510 confocal microscope.
Anti mouse igg alexa488
Manufactured by Thermo Fisher Scientific
Sourced in France, United States, United Kingdom
The Anti-mouse IgG-Alexa488 is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is designed to detect and visualize mouse immunoglobulin G (IgG) in various immunoassays and microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti hcv core, by Abcam (2 mentions)
T6793, by Merck Group (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Mounting reagent, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Dfc365fx, by Leica (1 mentions)
Dmi6000b af6000, by Leica (1 mentions)
24 well plate, by Corning (1 mentions)
Actin stain 555, by Cytoskeleton (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Mouse anti cd2, by Bio-Rad (1 mentions)
Rabbit anti cyce, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg alexa594, by Thermo Fisher Scientific (1 mentions)
Fluoview confocal microscope, by Olympus (1 mentions)
Anti ki67, by Agilent Technologies (1 mentions)
Anti glut1, by Thermo Fisher Scientific (1 mentions)
Opal 7 color fihc kit, by PerkinElmer (1 mentions)
Bx51 fl, by Olympus (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
8 protocols using anti mouse igg alexa488
1
Fluorescent Immunohistochemistry of Embryos
2
Dengue Virus Infection Assay in VERO Cells
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VERO cells (25 × 103 cells/well) were cultured on glass coverslips inside 24-well plates (Costar) overnight at 37°C and 5% CO2. Infections were performed with MOI of 0.1 with the DENV2 NGC strain for 1 h. After this period, supernatants containing the virus were discarded and the cells were incubated for 24 h in DMEM 5% FBS. Cells were fixed with ice-cold methanol for 5 min and the coverslips were blocked in PBS containing 1% BSA for 1 h at rt. Next, cells were incubated with pooled sera from the different mouse groups, or with the 4G2 mAb (as a positive control), during 1 h at rt, followed by another incubation with anti-mouse IgG-Alexa488 (1:2,000; Life technologies) in the same conditions. Nuclei were labeled with DAPI (1 μg/mL) and the images were acquired in a fluorescence microscope (Leica DMI6000B/AF6000, Buffalo Grove, IL, USA) coupled to a digital camera system (DFC 365 FX, Leica) and processed by the Leica Application Suite X (LAS X).
3
HCV Infection Quantification Assay
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WT and KO Huh7.5.1 cells were plated in 24 well plates and infected with HCV with a MOI of 0.1. 3 days post infection supernatant was collected and added to WT Huh7.5.1 cells in a 10-fold dilution series. After 3 days cells were fixed, stained with mouse-anti-HCV-core (Abcam ab2740) and anti-mouse-IgG-Alexa-488 (Life technologies) and fluorescent colonies were counted. Two independent experiments were performed with triplicate infections and one representative is shown.
4
Discriminating Tumor Tissue from Healthy in OncoCilAir
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To discriminate the cellular organization of tumor areas from healthy tissue in OncoCilAir™ model, the sample was fixed in 4% paraformaldehyde (PFA) for 30 min at RT, then permealized in 0.5% Triton X-100 for 15 min at RT. The cells were stained with either ethidium bromide (FT-25810A, Molecular Probes) or phalloidin (Actin-stain™ 555, PHDH1, Cytoskeleton) for 30 min at RT, for nuclear and F-actin staining respectively. Phalloidin stained the cell outline of the respiratory epithelium. Before microscopic observations, the tissues were placed on a glass slide, covered with an anti-fading mounting medium (H1000, VECTASHIELD®, Vector) and gently flattened using a large glass coverslip retained by adhesive tape.
The inserts were incubated with AGuIX® solution at 10 mM for 60 min to 72 h, in air/CO2 5%, 37 °C environment. The inserts were cryofixed using liquid nitrogen vapor, and sliced at 8 µm. A solution of 5% goat serum in PBS was used to block any unspecific sites. To discriminate ciliated and goblet cells, a beta IV tubulin antibody was used (#T6793, 1/500, Sigma-Aldrich, France) and anti-mouse IgG-Alexa488 (#A11029, 1/2000, Life Technology, France). Beta IV tubulin is a major constituent of microtubules in motile ciliary axonemes. The sections were incubated with 1 mM Hoechst solution before mounting with the coverslip.
The inserts were incubated with AGuIX® solution at 10 mM for 60 min to 72 h, in air/CO2 5%, 37 °C environment. The inserts were cryofixed using liquid nitrogen vapor, and sliced at 8 µm. A solution of 5% goat serum in PBS was used to block any unspecific sites. To discriminate ciliated and goblet cells, a beta IV tubulin antibody was used (#T6793, 1/500, Sigma-Aldrich, France) and anti-mouse IgG-Alexa488 (#A11029, 1/2000, Life Technology, France). Beta IV tubulin is a major constituent of microtubules in motile ciliary axonemes. The sections were incubated with 1 mM Hoechst solution before mounting with the coverslip.
5
HCV Infection Quantification Assay
6
Immunostaining of Drosophila Larval Discs
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For immunostaining, larval discs were dissected in PBS, fixed in 4% paraformaldehyde, blocked in 5% BSA, and incubated in primary antibodies overnight at 4°C, followed by secondary antibody incubation for 2 h at room temperature. The tissues were mounted with Vectashield mounting medium (Vectashield), and fluorescence images were acquired with a FluoView confocal microscope (Olympus). Primary antibodies used were rabbit anti-phospho-histone3 (Santa Cruz Biotechnology, Santa Cruz, CA, United States; 1:200), mouse anti-CD2 (AbD Serotec, 1:200), rabbit anti-CycE (Santa Cruz Biotechnology, 1:200), and goat anti-Diap1 (Santa Cruz Biotechnology, 1:200); and secondary antibodies were anti-mouse IgG Alexa 488, anti-rabbit IgG Alexa 594, and anti-goat IgG Alexa 594 (Life Technologies, 1:200).
7
Multimarker Fluorescence Immunostaining in Glioblastoma
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Fluorescence double immunostaining of GLUT1 and other markers (HIF1α, CD34, and Ki67) in tumor tissues was performed. Double staining was performed in a glioblastoma sample with antiGLUT1 (1:100, Thermo Fisher, UK), antiKi67 (1:100, Dako, Glostrup, Denmark), and antiCD34 (1:100, Leica, Milton Keynes, UK) antibodies followed by addition of antirabbit IgG-Texas Red (TR; sc-2780, Santa Cruz Biotechnology, Santa Cruz, CA), and antimouse IgG-Alexa488 (Life Technologies, Carlsbad, CA). Assessment of staining colocalization was performed using the OPAL 7-color fIHC Kit (Perkin Elmer, Waltham, MA) according to the manufacturer’s protocols. Fluorescence was measured using a fluorescent microscope (BX51FL, Olympus, Tokyo, Japan) and a charged-coupled device camera (DP71, Olympus).
8
Confocal Imaging of Regenerated Limb Nerves
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Large males were amputated at the distal part of the carpus of T4 and T5 limbs, as described earlier. After the first molt following regeneration they were anesthetized and fixed at room temperature for 10 minutes in 3.6% formaldehyde in ASW. The carpus and propodus podomeres of regenerated and unamputated control limbs were dissected and cut in half to improve antibody penetration. The samples were re-fixed for 15 minutes in 3.6% formaldehyde in PBS, washed for 1 hour in PBS with 0.1% Triton X-100 (PTx), and incubated for 1 hour at room temperature in PBS with 0.1% Triton X-100, 0.1% sodium deoxycholate, 5% bovine serum albumin and 0.5% normal goat serum (PAXD1). The samples were then incubated for 3 days at 4°C, with 1:1000 dilution of mouse monoclonal 6-11B-1 antibody for acetylated tubulin (Sigma T6793, RRID: AB_477585) in PAXD1, and washed overnight with PTx at 4°C. The samples were incubated with the secondary antibody (1:1000 dilution of anti-mouse IgG Alexa 488 (Life Technologies A11001, RRID: AB_2534069) in PAXD1 for 3 days at 4°C. Then the samples were washed overnight in PTx at 4°C and mounted in Vectashield mounting medium (Vector Labs H-1000). The samples were imaged on a Zeiss LSM 800 laser scanning confocal microscope.
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