Postmortem human brain tissue was obtained from the Neuropathology Core of the Alzheimer’s Disease Research Center (ADRC) at the University of Pittsburgh. The subjects’ information is listed in (Supp Table S1 ). IHC for formalin fixed paraffin embedded human brain sections were performed as described previously19 (link),20 (link). Briefly, after deparaffinization, the sections were subjected to heat-induced antigen retrieval process (Citra buffer, pH6.0, 99°C 20min), then blocked in blocking buffer (5% donkey normal serum, 1% BSA with 0.3% Triton X-100 in PBS) for 1 hour at room temperature. The sections were incubated in primary antibodies, Anti-AHR (1:500, NSJ Bioreagents) and anti-Iba1 (1:50, Fujifilm), and then detected by secondary antibodies, Alexa Fluor 488-conjugated Donkey anti-Rabbit and Alexa Fluor 647-conjugated Donkey anti-mouse (1:200, Jackson ImmunoResearch).
Alexa fluor 488 conjugated donkey anti rabbit
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor 488-conjugated donkey anti-rabbit is a secondary antibody used in immunodetection techniques. It is conjugated to the Alexa Fluor 488 fluorescent dye and specifically binds to rabbit primary antibodies, allowing for the detection and visualization of target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Cy3 conjugated donkey anti mouse, by Jackson ImmunoResearch (3 mentions)
Dapi fluoromount g, by Southern Biotech (3 mentions)
Alexa fluor 647 conjugated donkey anti mouse, by Jackson ImmunoResearch (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Rabbit anti foxp1, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Anti calreticulin, by Cell Signaling Technology (1 mentions)
Anti cytochrome c, by Santa Cruz Biotechnology (1 mentions)
Anti apol1, by Merck Group (1 mentions)
Hpa018885, by Merck Group (1 mentions)
Anti apol1 5.17d12, by Genentech (1 mentions)
T5168, by Merck Group (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Sc 13156, by Santa Cruz Biotechnology (1 mentions)
To pro 3, by Thermo Fisher Scientific (1 mentions)
Anti laminin, by Merck Group (1 mentions)
Axioplan2 epifluorescent microscope, by Zeiss (1 mentions)
Anti pax7, by Santa Cruz Biotechnology (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Orca er digital camera, by Hamamatsu Photonics (1 mentions)
12 protocols using alexa fluor 488 conjugated donkey anti rabbit
1
Immunohistochemical analysis of AHR and Iba1 in human brain
2
Immunostaining of Pancreatic Tissues
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Paraffin-embedded mouse and human pancreas sections were processed and stained as previously described (human donor information is provided in Table S2 ) [21] (link). Sections were stained using primary antibodies against somatostatin (Santa Cruz Biotechnology sc-7819: 1:250), TALK-1 (Novus Biologicals #NBP1-83071; 1:175) or TALK-1a (Antibody Verify AAS72353C; 1:250), glucagon (Proteintech #15954-I-AP: 1:500), and the endoplasmic reticulum marker GRP94 (Novus Biologicals #NB300-619; 1:100); secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rabbit (Jackson Immunoresearch #711-546-152; 1:300), DyLight 650-conjugated donkey anti-goat (Thermo Fisher #SA5-10089; 1:250), and Cy3-conjugated donkey anti-mouse (Jackson Immunoresearch #715-166-150; 1:500).
3
Retrograde Viral Tracing of RMTg Inputs
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Subjects were deeply anesthetized by inhaled isoflurane and transcardially perfused using 0.9% saline followed by 10% formalin. Brains were removed and stored in 10% formalin overnight before being transferred to 20% sucrose with 0.05% sodium azide for cryoprotection. Tissue was collected in 40μm-thick sections on a cryostat or freezing microtome, and floating sections were processed using immunohistochemistry to verify virus expression in the RMTg and accurate placement of cannulae or optical fibers in the VTA. Tissue was incubated overnight in PBS with 0.25% Triton X-100 (Sigma-Aldrich) and primary antibody for rabbit anti-GFP (1:50K, Abcam), mouse anti-tyrosine hydroxylase (TH; 1:10K, Millipore Inc.), or rabbit anti-FoxP1 (1:20K, Abcam). Fluorescence was visualized using 30-min secondary incubation in either Alexa Fluor 488-conjugated donkey anti-rabbit or Cy3-conjugated donkey anti-mouse secondary (1:1000, Jackson Immunoresearch). Following each incubation step, tissue was rinsed 3X in PBS at 1min/wash. Data were included from animals showing GFP+ cells clustered bilaterally in the region corresponding to the FoxP1-positive RMTg [15 (link)], with dense labelling of axon terminals in the TH-positive VTA.
4
Western Blot Antibody Validation
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Anti-APOL1 (1:2500 HPA018885, Sigma, St. Louis, MO, USA); Anti-APOL1 5.17D12 (1 µg/mL, kindly provided by Genentech, San Francisco, CA, USA); Anti-APOL1 oligoclonal 3.7D6/3.1C1 (0.05 µg/mL, kindly provided by Genentech); Anti-Tubulin (1:5000 T5168, Sigma, St. Louis, MO, USA); Anti-LC3 (1:1000, PM036, MBL, Chicago, MA, USA); Anti-Calreticulin (1:1000, 12238, Cell Signaling, Danvers, MA, USA); anti-Cytochrome C (1:1000, sc-13156, Santa Cruz, Dallas, TX, USA); Anti-Calnexin (1:2000, ADI-SPA-860 Enzo, New York, NY, USA); Anti-SDHB (Succinate Dehydrogenase Complex Iron Sulfur Subunit B, 1:2500, LS C497529, LSbio); Anti-rabbit HRP (1:20,000; 111-035-144, Jackson ImmunoResearch, Baltimore, PN, USA); Anti-mouse HRP (1:10,000; 115-035-166, Jackson ImmunoResearch); Alexa Fluor 488 conjugated Donkey anti Rabbit (1:600; 711-545-152, Jackson ImmunoResearch).
5
Immunostaining of Tibialis Anterior Muscle Sections
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Tibialis anterior (TA) muscle sections were prepared for staining essentially as previously described6 (link),48 (link). For OSMR staining, whole TA muscles were first fixed with 0.5% paraformaldehyde/PBS for 2 h at 4 C, followed by incubation in 20% sucrose/PBS overnight at 4 C. 10 μM sections were cut for all stainings. Antibodies were: anti-laminin (Millipore, catalog #05−206, 1:250), anti-GFP (Invitrogen, catalog #A11122, 1:200), anti-OSMR (R&D, catalog #AF665, 1:200), anti-Pax7 (Santa Cruz Biotechnology, catalog #sc81648, 1:50; or Developmental Studies Hybridoma Bank, 2 μg/mL final concentration), AlexaFluor 594–conjugated donkey anti-rat IgG1 and AlexaFluor 488–conjugated donkey anti-rabbit (Jackson ImmunoResearch, catalog # 712-585-150 and 711-545-152 respectively, 1:200 each). Nuclei were counterstained with either DAPI (Invitrogen) or TO-PRO-3 (Invitrogen). Images were acquired with an AxioPlan2 epifluorescent microscope (Carl Zeiss) with ORCA-ER digital camera (Hamamatsu Photonics).
6
Immunofluorescence of YTHDC2 Localization
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Immunofluorescence was essentially performed as previously described (Warda et al. 2016 (link)). In brief, HeLa cells or HEK293 cells expressing YTHDC2-Flag were grown on coverslips and fixed using 4% paraformaldehyde in PBS for 10 min at RT. Cells were permeabilized using 0.1% triton-X-100 in PBS for 15 min before blocking with 10% FCS and 0.1% triton-100 in PBS for 1 h at room temperature. Cells were then incubated with an anti-YTHDC2 antibody (Sigma HPA037364) or an anti-Flag antibody (Sigma F3165) in 10% FCS and PBS for 2 h at room temperature, washed thoroughly and then incubated with a secondary antibody (Alexa Fluor 488-conjugated donkey anti-rabbit; Jackson ImmunoResearch) for a further 2 h. After further washing steps coverslips were mounted using medium containing DAPI (Vectashield; Vector Laboratories). Finally, cells were analyzed by confocal microscopy using a ConfoCor2 microscope (Carl Zeiss).
7
Double Immunofluorescence Analysis of TLR2, TLR4, and Iba1 in Rat Brain
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Two SD rats of the control group were randomly selected for double immunofluorescence analysis. The brain sections were blocked for 10 min in PBS containing 10% bovine serum albumin (Sigma, USA). The sections were then incubated overnight at 4°C with primary antibodies [1 : 100 rabbit polyclonal anti-TLR2 (OriGene-Antibody, USA), 1 : 100 mouse monoclonal anti-TLR4 (Abcam, USA), and 1 : 500 goat polyclonal anti-Iba1 (Abcam, USA)]. Further, the sections were incubated with secondary antibodies [1 : 800 Alexa Fluor 488-conjugated donkey anti-rabbit, 1 : 800 Alexa Fluor 488-conjugated donkey anti-mouse, and 1 : 800 Cy™ 3-conjugated donkey anti-goat (Jackson ImmunoResearch Lab. Inc., USA)] for 1 h at room temperature. Each of the abovementioned steps was followed by three 3-min rinses in 0.01% Tween 20/PBS. At the end of the procedure, the sections were covered with coverslip using a mounting medium (SIGMA, USA) containing DAPI to counterstain DNA in the nuclei and dried overnight. The confocal images were captured using a laser-scanning confocal microscope (Leica TCS SP2, Germany).
8
Viral RNA Detection in Adipocytes and Macrophages
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Cells were fixed in 4% (v/v) paraformaldehyde (PFA) in PBS at 37 °C for 30 min, permeabilized with PBS containing 0.1% (w/v) Triton-X and blocked with 3% (v/v) BSA in PBS at RT for 30 min. Adipocyte cytoplasm was stained with wheat germ agglutinin conjugated with Alexa Fluor® 555 (W32464, Thermo Fisher Scientific) according to the manufacturer’s instruction before fixation. Adipocytes and macrophages were both incubated with mouse anti-dsRNA monoclonal antibody J2 (1:500; RNT-SCI-10010200, Jena Bioscience, Jena, Germany) or rabbit anti-dsRNA monoclonal antibody J2 (1:500, Kf-Ab01299-23.0, kerafast, Boston, MA, USA) to detect viral RNA. Additionally, macrophages were stained with mouse anti-CD68 monoclonal antibody (1:200; 14-0688-82, Invitrogen). Both primary antibodies were diluted in 3% (v/v) BSA in PBS incubated at 4 °C overnight. Secondary antibodies Alexa Fluor® 488-conjugated donkey anti-rabbit (711-545-152, Jackson ImmunoResearch) and Cy-3-conjugated donkey anti-mouse (715-165-150, Jackson ImmunoResearch) were each applied at 1:500 dilution. Cells were mounted with DAPI Fluoromount-G (Southern Biotech) and examined with an AxioObserver Z.1 microscope (Carl Zeiss AG).
9
Immunofluorescence Staining of FFPE Tissues
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For formalin-fixed paraffin-embedded tissues, mounted sections were dewaxed by xylene, rehydrated, and blocked. The sections were then incubated with primary antibodies for Histone H3 (1∶200, ab5103, Abcam, Cambridge, UK) overnight at 4 ℃. Then, the sections were washed and stained with fluorophore-conjugated secondary antibodies (Alexa Fluor 488-conjugated donkey anti-rabbit, 711-545-152, Jackson ImmunoResearch, West Grove, PA, USA) for 2h at room temperature. The secondary antibody used was. After washing 3 times with PBS, the samples were sealed with DAPI Fluoromount-G (0100-20, Southern Biotech, Birmingham, AL, USA) and were detected under fluorescence microscopy (LEICA DM2500) for morphologic details of the immunofluorescence staining. The examination was carried out blind.
10
Immunofluorescence Staining of Protein Complexes
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After fixation, beads were washed in AbDil (20 mM Tris-HCl, pH 7.4, 150 mM NaCl with 0.1% Triton X-100, and 2% bovine serum albumin), pipetted onto poly(l -lysine)-coated coverslips and allowed to adhere for ≥30 min. Coverslips were stained in primary antibody diluted in AbDil for ≥20 min and then washed in AbDil. Primary antibodies used were 2 µg/ml FLAG (F7425 [rabbit] and F1804 [mouse], both obtained from Sigma-Aldrich), 0.25 µg/ml Myc (4A6; EMD Millipore) 1 µg/ml xCENP-C (rabbit, raised and purified against xCENP-C207–296; Milks et al., 2009 (link)), and 1.5 µg/ml xM18BP1 (rabbit, raised against GST-xM18BP1-2 amino acids 161–415 and purified against xM18BP1-1161–375; Moree et al., 2011 (link)). Coverslips were then stained in secondary antibodies diluted in AbDil for ≥20 min, and then washed in AbDil. Secondary antibodies used were Alexa Fluor 488–conjugated donkey anti–rabbit (Jackson ImmunoResearch Laboratories, Inc.), Alexa Fluor 647–conjugated donkey anti–rabbit (Jackson ImmunoResearch Laboratories, Inc.), and Alexa Fluor 647–conjugated goat anti–mouse (Life Technologies) all at 2 µg/ml. Coverslips were washed in PBST and PBS, gently blotted with filter paper, and mounted in 90% glycerol, 10 mM Tris, pH 8.8, 0.5% p-phenylenediamine. Coverslips were sealed to a slide with clear nail polish.
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