Frozen wound tissues were cold acetone-fixed, washed in PBS-Tween20 (PBST) and incubated in normal goat serum blocking solution containing 2% goat serum, 1% BSA, 0.1% cold fish skin gelatin, 0.1% Triton X-100, 0.05% Tween-20, 0.05% sodium azide, and 0.01M PBS (pH 7.2) for 1h. For labeling, the single primary antibody or antibody mixes were applied for 16h at 4°C. After washing (5×) in PBST, slides were incubated with the secondary antibodies (Texas Red-conjugated anti-rabbit IgG and Alexafluor488-conjugated anti-rat IgG; Invitrogen, 1:500 dilution each) for 30 min, followed by washing (5×) in PBST. DAPI was used as counterstain (1 mg per ml). Fluorescence images were acquired using a Leica DM4000B microscope and processed with Photoshop CS2 (Adobe). Primary antibodies used for immunofluorescence staining are followed: anti-Ly6G rat mAb (1A8; BD Pharmingen, 1:100 dilution), anti-CD68 rat mAb (FA-11; Abcam, 1:100 dilution), anti-CD68 rabbit polyclonal antibody (Abcam, 1:200 dilution), anti-CD31 rat mAb (MEC 7.46; Abcam, 1:200 dilution), anti-NG2 rabbit polyclonal antibody (AB5320; Millipore, 1:100 dilution), anti-desmin rabbit polyclonal antibody (Abcam, 1:200 dilution), anti-CK14 mouse mAb (LL002; Abcam, 1:200 dilution), anti-vimentin rabbit polyclonal antibody (Abcam, 1:200 dilution), and anti-CCN1 rabbit polyclonal antibody (from the house, 1:200 dilution).
Alexa fluor 488 conjugated anti rat igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-conjugated anti-rat IgG is a secondary antibody that binds to rat immunoglobulin G (IgG) and is labeled with the fluorescent dye Alexa Fluor 488. This antibody can be used for detecting and visualizing rat IgG in various immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 568 conjugated anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Lsm 700 confocal microscope, by Zeiss (3 mentions)
Ab5320, by Merck Group (2 mentions)
Texas red conjugated anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Oct compound, by Sakura Finetek (2 mentions)
Alexa fluor 555 conjugated anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 546 conjugated anti rat igg, by Thermo Fisher Scientific (2 mentions)
Anti ty1, by Thermo Fisher Scientific (2 mentions)
Anti ha, by Roche (2 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (2 mentions)
Prolong antifade medium, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Anti foxp3 fjk 16s, by Thermo Fisher Scientific (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Zombie red, by BioLegend (1 mentions)
Anti nfat2 7a6, by BioLegend (1 mentions)
Alexa fluor 633 conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
23 protocols using alexa fluor 488 conjugated anti rat igg
1
Immunofluorescence Staining of Wound Tissue
2
Quantifying NFAT Nuclear Localization
3
Visualizing Intracellular TLR7 Localization in pDCs
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Primary human pDCs were purified from PBMCs using Diamond Plasmacytoid Dendritic Cell Isolation Kit II (Miltenyi Biotec), and purity was always >90%. PDCs were treated with IFN-α for 2 h. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and then treated with Image-iT FX Signal Enhancer (ThermoFischer) for preventing nonspecific staining. Cells were stained with rabbit anti-TLR7 (polyclonal: Novus Biologicals), mouse anti-EEA1 (1G11: Abcam), mouse anti-Rab7 (Rab7-117: Abcam), rat anti-LAMP-1 (1D4B: Abcam) and goat anti-BDCA2 (polyclonal: R&D systems) as primary Abs and subsequently stained with AlexaFluor488-conjugated anti-rat IgG, AlexaFluor594-conjugated anti-rabbit IgG and AlexaFluor647-conjugated anti-goat IgG (all from Invitrogen) as secondary Abs. Cells were spun onto a microscope slide using the Shandon Cytospin 4 (ThermoFischer) and mounted with Fluoromount/Plus (Diagnostic Biosystems). All samples were visualized using FM10i confocal laser scanning microscope (Olympus), and images were captured and analyzed using FV10-ASW viewer (Olympus). PDCs were identified as BDCA2 positive cells. Pearson's coefficient was calculated for analysis of the co-localization of TLR7 and endosomal markers (EEA1, Rab7, and LAMP1).
4
Immunofluorescence Staining of Wound Tissue
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Frozen wound tissues were cold acetone-fixed, washed in PBS-Tween20 (PBST) and incubated in normal goat serum blocking solution containing 2% goat serum, 1% BSA, 0.1% cold fish skin gelatin, 0.1% Triton X-100, 0.05% Tween-20, 0.05% sodium azide, and 0.01M PBS (pH 7.2) for 1h. For labeling, the single primary antibody or antibody mixes were applied for 16h at 4°C. After washing (5×) in PBST, slides were incubated with the secondary antibodies (Texas Red-conjugated anti-rabbit IgG and Alexafluor488-conjugated anti-rat IgG; Invitrogen, 1:500 dilution each) for 30 min, followed by washing (5×) in PBST. DAPI was used as counterstain (1 mg per ml). Fluorescence images were acquired using a Leica DM4000B microscope and processed with Photoshop CS2 (Adobe). Primary antibodies used for immunofluorescence staining are followed: anti-Ly6G rat mAb (1A8; BD Pharmingen, 1:100 dilution), anti-CD68 rat mAb (FA-11; Abcam, 1:100 dilution), anti-CD68 rabbit polyclonal antibody (Abcam, 1:200 dilution), anti-CD31 rat mAb (MEC 7.46; Abcam, 1:200 dilution), anti-NG2 rabbit polyclonal antibody (AB5320; Millipore, 1:100 dilution), anti-desmin rabbit polyclonal antibody (Abcam, 1:200 dilution), anti-CK14 mouse mAb (LL002; Abcam, 1:200 dilution), anti-vimentin rabbit polyclonal antibody (Abcam, 1:200 dilution), and anti-CCN1 rabbit polyclonal antibody (from the house, 1:200 dilution).
5
F4/80-Based Immunohistochemistry of WAT
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White adipose tissues (WAT) were isolated from around the epididymis and embedded in paraffin after fixed in 4% paraformaldehyde overnight, dehydrated with ethanol solutions, and transparented with Xylene. Four micron paraffin-embedded WAT sections were deparaffined, rehydrated and endogenous peroxidase quenched with 3% hydrogen peroxide, and microwaved for 15 min (with low heat) in citrate buffer (10 mM sodium citrate, pH 6) to repair antigen, and were then incubated for 60 min in blocking buffer [5% (v/v) goat normal serum (Millipore Bioscience Research Reagents) in PBS containing 0.3% (v/v) Triton X-100 (Sigma)] at room temperature (RT) and subsequently stained with anti-mouse F4/80 (Abcam,ab6640,1:100) antibody for overnight at 4°C. After washing the slides 3 times with 1% BSA in PBS, sections were treated with secondary antibody (alexa fluor 488 conjugated anti-Rat IgG, Invitrogen, A-2121, 1:600) for 1 h at RT. The sections were further washed and the nucleus was counter-stained with 4, 6-diamidino-2-phenylindole (DAPI) diluted to a concentration of 2 μg/ml in PBS (1:500) for 5 minutes. Sections were observed under Olympus fluorescence microscope using 20X objective. The Image-Pro software was used for image acquisition.
6
Immunohistochemical Analysis of Lymphocyte Subsets
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Lung tissue sections were deparaffinized in xylenes and were rehydrated by passage through serial dilutions of ethanol in distilled water. Heat-induced antigen retrieval was performed in an antigen retrieval solution (ImmuoActive; Matsunami Glass Ind., Ltd., Kishiwada, Japan) with the use of a microwave, twice, for 5 min. The sections were incubated with Blocking One Histo (Nacalai Tesque) in order to block non-specific reactions. Anti-mouse B220 (eBioscience), anti-mouse CD3 (Cell Signaling Technology) monoclonal antibodies (mAbs), and anti-mouse CD23 (Boster Biological Technology) polyclonal Ab were applied to the sections overnight, at 4°C. After washing with phosphate-buffered saline (PBS), the sections were incubated with an Alexa Fluor 488-conjugated anti-rat IgG (Invitrogen) or an Alexa Fluor 568-conjugated anti-rabbit IgG (Invitrogen) Ab. After washing thrice with PBS, the nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Waltham, MA), and the sections were observed under an optical microscope (ZEISS Axio Observer 7; Carl Zeiss) and were analyzed through the use of ZEN3.2 blue edition (Carl Zeiss).
7
Histological Analysis of Tumor Tissues
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Tumor tissues were harvested from mice at 6 days after final treatment and fixed in 10% formalin, processed for paraffin embedding, and cut into 5-mm sections. Sections were stained with hematoxylin and eosin (H&E) and examined by light microscopy. Tumor sections were also immunostained with rabbit anti-Ad E1A polyclonal Ab (Santa Cruz Biotechnology) to assess viral replication and spreading. Slides were counterstained with Meyer's hematoxylin. To detect lymphocytes, tumor tissues were dehydrated with 30% sucrose at 4°C, frozen in OCT compound (Sakura Finetec, Torrance, CA), and cut into 10-mm sections. Cryosections were incubated with rat anti-mouse CD4 monoclonal Ab (BD Biosciences), rat anti-mouse CD8 monoclonal Ab, or rabbit anti-Foxp3 monoclonal Ab (Abcam, Cambridge, UK). After incubating with primary Ab at 4°C overnight, sections were incubated with Alexa Fluor 488-conjugated anti-rat IgG (Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 568-conjugated anti-rabbit IgG as secondary Abs for 1 hr. For counterstaining, samples were incubated with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and semiquantitatively analyzed by ImageJ software.
8
Flow Cytometric Analysis of Histone Modifications
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For live cell analysis, dispersed ES cells were washed with PBS and suspended with FACS buffer (PBS/2% fetal bovine serum/0.02% NaN3), and the EmGFP signals were analyzed by FACSCantoII or LSRFortessa X-20 (Beckton Dickinson) at FITC channel. For immunostaining analysis, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with FACS buffer containing 0.5% Tween 20 were incubated with anti-H3K27ac or anti-FLAG M2, and with anti-GFP antibodies at 4 °C for overnight. The cells were washed with FACS buffer containing 0.1% Tween 20 three times followed by incubation with secondary antibodies, PE-conjugated anti-mouse IgG (BD Pharmingen), and Alexa Fluor 488-conjugated anti-rat IgG (Invitrogen), for 30 min. Washed samples were analyzed by LSRFortessa X-20 (Beckton Dickinson). Data was analyzed by using BD FACSDiva software (Becton Dickinson). Each analysis was done in two to five independent experiments with biological triplicates for each set of experiment.
9
Immunohistochemical Analysis of CD31 and GFP in Muscle Tissue
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The procedure for tissue preparation and staining was previously reported [18 (link)]. For immunohistochemistry, an anti-CD31 antibody was used for staining and a biotin-conjugated polyclonal anti-rat IgG (Agilent Technologies, CA, USA) was used as the secondary antibody. Biotinylated secondary antibodies were developed using ABC kits (Vector Laboratories, CA, USA). Samples were visualized using a Canon EOS kiss X7 for the low-powered field, and a Leica DMi8 for the high-powered field. Images were processed with the Leica application suite (Leica Microsystems, Wetzlar, Germany), Adobe Photoshop CC software (Adobe Systems, CA, USA). For confocal microscopic images, sections were prepared as previously reported [19 (link)]. In brief, hind limb muscles were excised, fixed in 4% PFA/PBS, washed with PBS, embedded in OCT compound (Sakura Finetek, Tokyo, Japan), and sectioned (60 μm). The sections were stained with an anti-CD31 monoclonal antibody and anti-GFP polyclonal antibody (MBL, MA, USA). Alexa Fluor 488-conjugated anti-rat IgG and Alexa Fluor 647-conjugated anti-rabbit IgG (Invitrogen, CA, USA) were used as secondary antibodies. The sections were visualized using a Leica TCS SP5 confocal microscope and processed with the Leica application suite and Adobe Photoshop CC software. All images shown are representative of more than two independent experiments.
10
Quantifying Muscle Macrophage Polarization
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