WT- and 3Tg-iAstro cells, grown on 13 mm glass coverslips, were fixed in 4% formaldehyde, permeabilized (7 min in 0.1% Triton X-100 in phosphate-buffered saline (PBS)) and immunoprobed with an appropriate primary antibody (diluted in PBS supplemented with 1% gelatin) for 1 h at 37 °C. After 3 times washing in PBS, an Alexa-conjugated secondary antibody (1:300 in PBS supplemented with 1% gelatin) was applied for 1 h at room temperature (RT). The following primary antibodies were used: AQP4 (Alomone Labs, Cat. No. 249-323), Aldh1l1 (Abcam, Cat. No. Ab190298), GS (Abcam, Cat. No. Ab73593), GFAP (Chemicon International, Cat. No. CBL411) and GLT-1 (Alomone labs, Cat. No. AGC-022). Secondary antibodies were as follows: Alexa Fluor 488 anti-mouse IgG, Alexa Fluor 555 anti-rabbit IgG (all secondary antibodies were from Molecular Probes, Life Technologies, Monza, Italy). Nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired by Zeiss 710 confocal laser scanning microscope equipped with EC Plan-Neofluar 40×/1.30 Oil DIC M27 objective and Zen software.
Alexa fluor 555 anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy
Alexa Fluor 555 anti-rabbit IgG is a secondary antibody conjugated with the Alexa Fluor 555 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays, such as immunofluorescence, flow cytometry, and Western blotting.
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Lab products found in correlation
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (13 mentions)
Anti mouse igg alexa fluor 555, by Thermo Fisher Scientific (3 mentions)
Anti ki67, by Cell Signaling Technology (2 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Alexa fluor 488 anti rat igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 anti goat igg, by Thermo Fisher Scientific (2 mentions)
Blocking one, by Nacalai Tesque (2 mentions)
Ec plan neofluar 40 1.30 oil dic m27 objective, by Zeiss (1 mentions)
710 laser scanning confocal microscope, by Zeiss (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
Aldh1l1, by Abcam (1 mentions)
Anti puromycin, by Merck Group (1 mentions)
Nb100 1869, by Novus Biologicals (1 mentions)
Fv500, by Olympus (1 mentions)
Labtek permanox chamber slides, by Thermo Fisher Scientific (1 mentions)
Dmr microscope, by Leica (1 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 phalloidin, by Thermo Fisher Scientific (1 mentions)
Evos m5000 imaging system, by Thermo Fisher Scientific (1 mentions)
30 protocols using alexa fluor 555 anti rabbit igg
1
Immunostaining of Astrocytic Markers
2
Immunofluorescence Analysis of Astrocytes
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Primary mouse hippocampal and foetal human astrocytes, grown on 13 mm glass coverslips and treated as indicated, were fixed in 4% formaldehyde and 4% sucrose, permeabilized (7 min in 0.1% Triton X-100 in phosphate-buffered saline (PBS)), blocked in 1% gelatin, and immunoprobed with an appropriate primary antibody (diluted in PBS supplemented with 1% gelatine) over night at 4 °C. After 3 times washing in PBS, an Alexa-conjugated secondary antibody (1:300 in PBS supplemented with 1% gelatine) was applied for 1 h at room temperature (RT). The following primary antibodies were used: anti-GLAST (rabbit, 1:100, Cat. NB100-1869, Novusbio, Abingdon, UK), anti-puromycin (1:200, Millipore, Cat. MABE343). Secondary antibodies were as follows: Alexa Fluor 488 anti-mouse IgG, Alexa Fluor 555 anti-rabbit IgG (all secondary antibodies were from Molecular Probes, Life Technologies, Monza, Italy). Nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using an FV-500 Olympus (Tokyo, Japan) laser confocal scanning system with a 60X oil immersion objective. Immunofluorescence signal intensity per cell was measured with NIH ImageJ software v1.52p and was calculated as corrected total cell fluorescence (CTCF) = Integrated Density—(Area of selected cell X Mean fluorescence of background readings).
3
Immunofluorescence Analysis of Stem Cell Markers
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Freshly isolated MPCs, P2-MSCs, and HUVECs were plated in two-well Lab-Tek™ Permanox chamber slides (Thermo Scientific, Rochester, NY, USA). Slides were fixed for 15 min in 4% paraformaldehyde at room temperature and subsequently permeabilized with 0.5% Triton X-100 for 30 min. Immunofluorescence was carried out using mouse monoclonal anti-human Nestin (Abcam, Cambridge, UK) and rabbit polyclonal anti-human von Willebrand factor antibodies (Abcam). Positive stain was revealed by the goat anti-mouse SFX kit (Thermo Scientific), according to the manufacturer’s instructions using AlexaFluor®-488 anti-mouse IgG and AlexaFluor®-555 anti-rabbit IgG (Thermo Scientific). F-Actin was detected by AlexaFluor®-555 Phalloidin (Thermo Scientific). Slides were mounted in Prolong® Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Thermo Scientific) for nuclei detection. Pictures were taken and combined using a standard fluorescence DMR Leica microscope (Leica, Wetzlar, Germany) equipped with Leica CW4000 image software (Leica).
4
Immunohistochemical Analysis of Hair Follicle Tissue
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Skin tissue sample preparations and treatments were described previously [3 (link)]. Paraffin-embedded hair follicles were cut out to a thickness of 5 micrometers. Immunohistochemical analysis was performed as previously described [3 (link)]. The tissues were first stained with primary antibodies (anti-Ki67, #12202, Cell Signaling Technology, Danvers, MA, USA; anti-β-catenin, #8480, Cell Signaling Technology; and anti-CD34, sc-74499, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then with secondary antibodies (Alexa Fluor 555 anti-rabbit IgG or Alexa Fluor 488 anti-mouse IgG, Thermo Fisher Scientific KK, Tokyo, Japan). After staining with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA), the tissue samples were observed with the EVOS M5000 Imaging System (Thermo Fisher Scientific).
5
Immunofluorescent Labeling of Bdnf-P2a-Gfp Mice
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Bdnf-P2a-Gfp mice were killed at P90 by pentobarbital injections transcardially perfused with ice-cold PBS and 4% PFA. Their brains were removed and postfixed at room temperature for 4 h before cryoprotecting in 30% w/v sucrose solution at 4°C overnight. The following day, brains were embedded in OCT (optimal cutting temperature) compound and sectioned at 40 μm using a cryostat. Sections were blocked in blocking solution (3% donkey serum and 4% BSA in PBS-T) for 1 h before incubating overnight with chicken anti-GFP (Abcam 13970, 1:1,000) and rabbit anti-Tmem119 (Abcam 209064, 1:500) or rabbit anti-NeuN (Abcam 177487). Sections were then washed three times for 10 min with PBS-T before incubating with Alexa Fluor 555 anti-rabbit IgG and Alexa Fluor 488 anti-chicken IgY secondary antibodies (1:500; Thermo Fisher Scientific) for 1 h at room temperature. After a final wash in PBS-T for 10 min, sections were incubated with DAPI diluted in PBS (1:4,000) for 20 min and mounted onto precoated polylysine slides (VWR) with Dako fluorescence mounting media. Two sections from two animals were analyzed.
6
Immunohistochemical Analysis of Hippocampal and Amygdalar Neurons
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Brains (n = 6/treatment group from different litters generated in Experiment 2) were processed for immunohistochemistry. Coronal cryostat sections (12 μm) including the hippocampus (CA1, CA2, CA3) and the basolateral amygdala were mounted as contiguous triplicates. Sections were fixed in cold methanol (−20 °C), blocked with 5% goat serum, 0.3% Triton X-100 in phosphate buffered saline (PBS) for 2h at 4 °C, followed by an overnight incubation at 4 °C in primary antibody against either parvalbumin (1:500, ab11427; abcam), MAP2 (1:500, ab32454; abcam), GABA Aα1 (1:500, ab33299; abcam), GABA Aα2 (1:500, ab193311; abcam) or GABA B1 (1:500, ab55051; abcam) in PBS with 1% bovine serum albumin (BSA), 0.3% Triton X-100. Sections were incubated with Alexa Fluor 555 anti-rabbit IgG, Alexa Fluor 488 anti-mouse IgG or Alexa Fluor 568 anti-mouse IgG secondary antibodies (Thermo Fisher Scientific) at 1:500 for 2h at 4 °C. Vectashield Mounting Medium with DAPI (Vector Laboratories, USA) was used to mount coverslips.
7
Characterization of Recombinant LN521 Coating
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Human recombinant LN521 (Biolamina, Sundbyberg, Sweden) was diluted to 20 µg/mL in acidic buffer (20 mM sodium acetate, pH 4.0) containing 1 mM CaCl2. Drops containing 10 μL were placed on glass coverslips and incubated for 12 h, after which they were fixed in either 4% paraformaldehyde for immunolabeling or in Karnowsky reagent (4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2) for 2 h for scanning electron microscopy (SEM). For immunolabeling, samples were incubated with rabbit polyclonal anti-LM antibody (1:50; Millipore-Sigma, St. Louis, MO, USA) and then with Alexa Fluor 555 anti-rabbit IgG (1:300, Thermo Fisher Scientific, Waltham, MA, USA). Confocal fluorescence images were obtained using a Zeiss Elyra PS1. For SEM, dehydrated samples dried with critical point drying were coated with a thin layer of gold sputter and visualized by using a JEOL JSM6300 scanning electron microscope. For transmission electron microscopy (TEM), 3 μL of polyLN521 were added to 300 mesh Cu-grids (Ted Pella Inc., Redding, CA, USA) that were previously glow-discharged for 180 s at 15 mA (Ted Pella, PELCO easiGlow™). After 30 min, the excess liquid was removed, and 1% uranyl acetate was added to samples for contrasting. Samples were analyzed using a 120-kV transmission electron microscope (Hitachi HT 7800, Japan).
8
Immunostaining for Endothelial Markers
9
Immunofluorescence Staining of Gastric Cancer Cells and Tissues
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For cells, glass chambers were treated with poly‐L‐lysine for 1 h, and then gastric cancer cells were grown into the chamber (FALCON, #354118), and cells were fixed with 4% PFA for 1 h at room temperature. For tissues, the tumor samples were embedded in paraffin and the samples were cut into 4 μm sections. The sections were dewaxed in xylene and then gradually dehydrated with gradient alcohol and excess wax was removed. Sections were subjected to antigen repair in Citrate solution (10 mM, pH 6.0) at 100°C for 20 min. Next, cells or slides were treated with 0.1% Triton X‐100 for permeabilization on ice for 5 min, followed by dressing with 5% BSA for 1 h at room temperature to reduce non‐specific staining. Cells or slides were incubated overnight at 4°C with primary antibodies (anti‐NF‐κB p65, Beyotime, #AN365, 1:200 dilution; anti‐Ki67, abcam, #ab15580l, 1:500 dilution; anti‐PCNA, Dako, #M0879, 1:500 dilution; anti‐CD206, Proteintech, #18704‐1‐AP, 1:200 dilution) followed by secondary antibodies (Alexa Fluor™ 555 anti‐rabbit IgG, ThermoFisher Scientific, #A‐31572, 1:500 dilution; Alexa Fluor™ 488 anti‐mouse IgG, ThermoFisher Scientific, #A‐11029) for 1 h at 37°C. Cell nuclei were stained with DAPI (Beyotime, #C1002).
10
Localization of P450 in Chum Salmon Brain
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IHC localization of P4507α was performed as described previously19 (link)21 (link)22 (link). In brief, chum salmon captured at their pre-spawning ground during upstream migration were terminated by decapitation. The brains were fixed in 4% (vol/vol) paraformaldehyde solution overnight, and they were soaked in a refrigerated 30% (vol/vol) sucrose solution in 0.1 M PB. Whole brains were frozen in OCT compound (Miles) and sectioned transversely at 20-μm thickness on a cryostat at –20 °C. After blocking nonspecific binding with 5% (vol/vol) normal goat serum and 1% BSA in PBS containing 0.5% Triton X-100, the sections were immersed overnight at 4 °C in 1:100 dilution of rabbit anti-salmon P4507α antibody. The sections were then incubated for 60 min with Alexa Fluor 555 anti-rabbit IgG (Thermo Fisher Scientific) at a dilution of 1:1,000. After washing, the sections were mounted with mounting medium and visualized by using a fluorescence microscope (Leica).
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