Adult B. malayi were mounted in Cryo-M-Bed (Bright Instruments), frozen on dry ice, and 5 μm sections cut using a Leica CM1510S cryotstat. Air dried sections were fixed in 100% acetone (10 min), washed twice with PBS (20 min), and stained in a humidified chamber with 1/100 dilution mouse anti-Bm-TPI sera (generated as above) in 1% FCS/PBS (1 hour at room temperature). Control sections were similarly treated with naïve mouse sera. Following extensive washing in PBS, sections were incubated with 1/100 goat anti-mouse IgG TRITC (Sigma) as above, washed in PBS, then mounted with Vectashield (Vector labs). Sections were analysed with an Olympus BX50 fluorescent microscope and Openlab software (PerkinElmer).
Goat anti mouse igg tritc
Manufactured by Merck Group
Sourced in United States
Goat anti-mouse IgG TRITC is a secondary antibody labeled with the fluorescent dye Tetramethylrhodamine (TRITC). It is designed for the detection and visualization of mouse immunoglobulin G (IgG) in various immunoassays and microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg fitc, by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Openlab software, by PerkinElmer (1 mentions)
P4417, by Merck Group (1 mentions)
P9416, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Propyl gallate, by Merck Group (1 mentions)
Dm2500 fluorescent microscope, by Leica (1 mentions)
Jb 4 resin, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Immobilon membrane, by Merck Group (1 mentions)
Mouse anti β tubulin antibody, by Merck Group (1 mentions)
Goat anti rabbit igg peroxidase, by Merck Group (1 mentions)
Mouse anti c myc antibody, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg fitc, by Merck Group (1 mentions)
Tnf α, by Sino Biological (1 mentions)
Rabbit anti iκβα antibody, by Merck Group (1 mentions)
Goat anti mouse igg peroxidase, by Merck Group (1 mentions)
8 protocols using goat anti mouse igg tritc
1
Immunostaining of B. malayi Cryosections
2
Immunofluorescence Staining of Worms
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The immunofluorescence staining of worms was performed as previously described [43 (link)]. Briefly, the collected worms were permeabilized by flash-freezing in liquid nitrogen. Fixation was carried out by sequential incubation in methanol and acetone at −20°C. The worms were blocked in PBS buffer (Sigma-Aldrich, P4417) containing 5% BSA (Sigma-Aldrich, SLCF3318) and 0.5% Triton X-100 (Sigma-Aldrich, 9036–19-5), then incubated with primary antibodies at 4°C overnight, followed by a series of incubation and washing steps in PBST (0.05% Tween 20 [Sigma-Aldrich, P9416]) buffer. The worms were incubated with secondary antibodies for 1 h, then extensively washed and mounted with anti-fade Fluorescence Mounting Medium (Sigma-Aldrich, F6057). Primary and secondary antibodies were used at the following concentrations: anti-IFB-2 (Developmental Studies Hybridoma Bank [DSHB], 528311; 1:20); anti-DLG-1 (DSHB, 2617529; 1:10); anti-FLAG (Sigma-Aldrich, F7425; 1:50); goat anti-mouse IgG-TRITC (Sigma-Aldrich, T5393; 1:100).
3
Immunofluorescence Staining of F. hepatica
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Adult F. hepatica were collected from the bile ducts of sheep immediately after slaughter at a local abattoir (Dungannon, Northern Ireland) . Worms were fixed for 4 h in 4% paraformaldehyde in 0.1 M PBS (Sigma-Aldrich) and subsequently embedded in JB-4 resin (Sigma-Aldrich) according to the manufacturer's instructions. Semi-thin (2 µm) sections were cut on a pyramitome and mounted on clean glass slides. For immunofluorescence, tissue sections were washed in PBS then incubated overnight in primary antibody in antibody diluent (AbD: PBS containing 0.2% (v/v) Triton X-100) at room temperature (18-21°C). For details of the primary antibodies, including their target peptides, working concentrations and source, see Supplementary Table 1. As a negative control, tissue sections were also incubated with pre-immune serum in AbD. The sections were then washed three times in AbD before incubation in an appropriate secondary antibody-fluorophore conjugate (goat anti-rabbit IgG-FITC or goat anti-mouse IgG-TRITC, Sigma-Aldrich) at a 1:200 dilution in AbD for 1 h at room temperature. Following four washes in PBS, the sections were mounted in glycerol containing 10% (v/v) PBS and 0.1 M propyl gallate (Sigma-Aldrich) then viewed under a Leica DM2500 fluorescent microscope.
4
Transendothelial Migration and Permeability Assay
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WJ-MSCs (ENG+/+ and ENG−/−) were placed on top of confluent HUVEC monolayers grown on transwell inserts. After 24 h, when the stem cells have transmigrated, FITC-albumin (1 mg/ml) was added to the apical chamber and the leaked tracer was quantified at the basal compartment (Figure 7 A). The data are expressed as the amount of leaked protein to the basal compartment in the two co-culture groups (HUVEC+ ENG−/− WJ-MSCs and HUVEC+ ENG+/+WJ-MSCs). For immunostaining, PKH67 green dye-labelled WJ-MSCs (ENG+/+ and ENG−/−) (Sigma Aldrich, U.K., catalog#PKH67GL) were placed on top of confluent HUVEC monolayers, and the co-culture bilayers were fixed and stained for VE-cadherin. After washes, cells were incubated for 2 h in the dark with goat anti-mouse IgG-TRITC (Sigma Aldrich, U.K., catalog# T5393) (1:100) and the continuous junctions were counted.
5
Evaluating Anti-cancer Mechanisms of KLT Injection
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All cell culture media, antibiotics, and trypsin were purchased from Gibco (Grand Island, NY, USA), and fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). Mouse anti-E-cadherin antibody, mouse anti-NF-κΒ p65 antibody, mouse anti-cyclin D1 antibody, mouse anti-c-myc antibody, and mouse anti-snail antibody were purchased from Santa Cruz Biotechnology (CA, USA). Rabbit anti-matrix metalloproteinase-9 (MMP-9) antibody, rabbit anti-IKKα antibody, rabbit anti-IκΒα antibody, rabbit anti-vimentin antibody, rabbit anti-Histone H3 antibody, mouse anti-β-tubulin antibody, goat anti-rabbit IgG-peroxidase, goat anti-mouse IgG-peroxidase, goat anti-mouse IgG FITC, goat anti-rabbit IgG FITC, goat anti-mouse IgG TRITC, DAPI and methyl thiazolyl tetrazolium (MTT), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Immobilon membranes were purchased from Merck Millipore (Bedford, MA, USA). ECL Plus substrate, bicinchoninic acid reagents, and RIPA lysis buffer were purchased from CWBio (Beijing, China). TNF-α was purchased from Sino biological Inc. (Beijing, China). KLT injection was purchased from Zhejiang Kanglaite Pharmaceutical Co., Ltd, (Zhejiang, China). Nuclear and cytoplasmic protein extraction kit, crystal violet were purchased from Beyotime (Shanghai, China).
6
Prostate Cancer Cell Proliferation Assay
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MIGR-hKCTD11-infected prostate cancer cell lines were cultured onto a 12 mm diameter glass. After 24 hrs, BrdU was added (BrdU labeling and detection kit, Boehringer Mannheim) according to the manufacturer's instructions. Cells were incubated for 8 and for 24 hours and then fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.25% Triton X in PBS, washed, and incubated with primary antibody (anti-GFP, Santa Cruz Biotechnology). After 1 hr, cells were washed and incubated with goat anti-rabbit Alexa Flour 488 (Santa Cruz Biotech) secondary antibody for 45 min. Cells were treated with 4% paraformaldehyde and then 2 N HCl. After three washes, cells were incubated with 1 : 10 primary anti-BrdU (BrdU labeling and detection kit, Boehringer Mannheim) and, afterward, with goat anti-mouse IgG TRITC (Sigma-Aldrich) secondary antibody. Hoechst was used for nuclear staining. Cells were analyzed by using a Zeiss Axioplan 2 (Carl Zeiss) fluorescence microscope.
7
Immunofluorescent Staining of Neurons
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Primary neuronal cells were fixed with 4% paraformaldehyde in PBS for 10 min and then washed with PBS. Cells were saturated with 0.3% Triton X-100 in 1% bovine serum albumin (BSA) in TBS-FCS 10% for 15 min, followed by three washes in TBS and incubated 2 h with rabbit anti-p-ERK (9101 1/200, Cell Signaling) and mouse anti-MAP2 (119942 1/250, Sigma) in TBS with 1% BSA, 10% FCS, and 0.3% Triton X-100. Then, cells were washed three times with TBS and incubated with the corresponding secondary antibodies, sheep anti-rabbit IgG FITC (F7512 1/500, Sigma) or goat anti-mouse IgG TRITC (T7657 1/100, Sigma) for 1 h in a wet and dark room. After three washes, cells were stained with bisBenzimide H33258 (B1155, Sigma) for 10 min and washed and mounted onto microscope slides with Fluoromount-G (0100-01, Southern Biotech). The costainings were observed using an inverted Zeiss Cell Observer 27 microscope with a 40× EC Plan Neofluar 40/0.75 numerical aperture objective (Carl Zeiss Co.). Images were processed with Zen software analyzed with ImageJ.
8
Sciatic Nerve Immunohistochemistry Protocol
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Four weeks after surgery, the sciatic nerves were removed, fixed in 4% paraformaldehyde for 12 hours, then put in 30% sucrose phosphate buffer for 24 hours until they sunk to the bottom of the container. A cryostat microtome was then used to cut 20–30 μm thick cross sections. The primary antibodies mouse anti-S100 (1:500; Sigma, St. Louis, MO, USA), and anti-neurofilament 200 (1:80; Sigma) were applied overnight at 4°C. Secondary antibodies goat anti-rabbit IgG (FITC) and goat anti-mouse IgG (TRITC) (both Sigma) were applied for 1 hour at room temperature (He et al., 2016; Wang et al., 2016). Images were acquired using a laser confocal microscope (FV10i-oil, push around, Tokyo, Japan).
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