After thawing the cytoslides, a hydrophobic circle was drawn around the cell-containing area and 30 µL of PBS was applied to this area to rehydrate the preparation. After rehydration, approximately 60 µL of the antibody solution was applied to the cytopreparation, which was incubated overnight in a humidified chamber at 4 °C on a shaking platform. For detecting the large T-antigen, the affinity purified anti-SV40 large T-antigen-specific antibody (diluted 1:100) PAS 112036 was used (Invitrogen, Waltham, MA, USA), and for staining BK polyoma virus VP1, the monoclonal (diluted 1:30) MAB:33242 clone 4942 (Invitrogen) was used. The next morning, the cytopreparation was washed in PBS with constant agitation of the wash liquid. Then, the secondary antibody solution Alexa-Fluor 488 goat anti rabbit IgG (A11008) or Alexa-Fluor 594 goat anti mouse IgG (H+L) (A11032, Invitrogen) was applied to the labeled areas containing the cells. After 60 min incubation in the moist chamber, the slides were washed in PBS for 10 min with constant stirring. For nuclear DNA staining, a three-minute staining with DAPI was performed before washing. Finally, an embedding solution and a glass coverslip were applied before images were recorded using an Axiovert confocal microscope.
Alexafluor 488 goat anti rabbit igg a 11008
Manufactured by Thermo Fisher Scientific
Sourced in United States
AlexaFluor 488 goat anti-rabbit IgG (A-11008) is a fluorescently-labeled secondary antibody used for detecting and visualizing primary rabbit antibodies in various immunodetection applications. It is conjugated with the AlexaFluor 488 fluorescent dye, which emits green fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti mouse a11032, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
One glo tox luciferase reporter and cell viability assay kit, by Promega (1 mentions)
Cell culture reagents, by Avantor (1 mentions)
0.22 μm pore filter, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Celastrol, by Merck Group (1 mentions)
Phosphor akt, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Tamoxifen, by Bio-Techne (1 mentions)
Hoechst 33342, by RiboBio (1 mentions)
Phosphor mtor, by Cell Signaling Technology (1 mentions)
Lc3 1 2, by Cell Signaling Technology (1 mentions)
Hoechst 33342 nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Ab26278, by Abcam (1 mentions)
Pa3 900, by Thermo Fisher Scientific (1 mentions)
Percp efluor 710 conjugated anti mouse cd3 clone 17 a2, by Thermo Fisher Scientific (1 mentions)
11 protocols using alexafluor 488 goat anti rabbit igg a 11008
1
Fluorescent Immunolabeling of Viral Proteins
2
In Vitro Evaluation of Mannose-Targeted Nanocarriers
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Cell culture reagents and trypsin were purchased from VWR (Radnor, PA, USA) or Corning Cellgro (Manassas, VA, USA). Fetal Bovine Serum (FBS) was obtained from Atlanta Biologicals. Opti-MEM was purchased from GibcoTM (Life technologies, Carlsbad, CA, USA). mPEG-PCL (5000:20,000) and PEG-PCL conjugated with aminofluorescein were purchased from PolySciTech Inc. (West Lafayette, IN, USA). α-D-mannopyranosylphenyl isothiocyanate (MPITC) was purchased from Carbosynth Ltd. (Berkshire, UK). Simulated gastric fluid, simulated intestinal fluid, lipase were obtained from Fisher (Hampton, NH, USA). Anti-mannose receptor (CD206) antibody (ab64693) was acquired from Abcam (Cambridge, MA, USA), Alexa Fluor 488 goat anti-rabbit IgG (A11008) was purchased from Invitrogen (Carlsbad, CA, USA). ONE-GLO + Tox Luciferase Reporter and cell viability assay kit were from Promega (Madison, WI, USA). GFP quantification kit (ab235672) was purchased from Abcam (Cambridge, MA, USA).
3
Combination Therapy Potentiates Autophagy
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Tamoxifen was purchased from Tocris (Minneapolis, MN). Celastrol was purchased from Shanghai Hotmed Sciences Co. Ltd. (Shanghai, China), with 98% purity or higher. Dimethyl sulfoxide (DMSO) was purchased from Sigma (St. Louis, USA). Tamoxifen and Celastrol were dissolved in DMSO (0.1%, v/v, final concentration), then sterilized by a 0.22 μm pore filter (Merck Millipore, Bedford, USA). Antibodies were purchased from the following companies: LC3I/II, P62, phosphor-Akt, and phosphor-mTOR from Cell Signaling Technology (Danvers, UK); β-actin, anti-mouse-IgG, and anti-rabbit-IgG from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and Hoechst 33342 from Guangzhou Ribobio (Guangzhou, China). The Annexin V-FITC Apoptosis Detection Kit and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from KeyGEN Biology Co. (Nanjing, China). Alexa Fluor 488 goat anti-rabbit-IgG (A11008) was purchased from Invitrogen (Carlsbad, CA).
4
Immunofluorescent Staining of Gut Enteroendocrine Cells
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Colon tissues were fixed in 4% paraformaldehyde in PBS for 24 h and washed and dehydrated with ethanol. Paraffin-embedded sections (8 μm) were prepared. For staining, sections were deparaffinized and exposed to antigen unmasking in antigen retrieval 2100 using 10 mM sodium citrate buffer pH 6.0. After rinsing, sections were incubated in blocking buffer (10% goat serum, 1% bovine serum albumin and 0.1% Triton X-100 in PBS) for 1 h at room temperature. Sections were stained with anti-GLP-1 mouse monoclonal subtype IgG1 antibody (ab26278, Abcam, Cambridge, UK) diluted 1:400 or anti-peptide YY (PYY) chicken polyclonal antibody (ab15879, Abcam) diluted 1:800, and anti-INSL5 rabbit polyclonal antibody (G-035-40, Phoenix Pharmaceuticals, Burlingame, CA, USA) diluted 1:200 in blocking buffer overnight at 4 °C. Primary antibodies were targeted with immunofluorescent dye labeled secondary antibodies Alexa Fluor 568 anti-mouse IgG1 (γ1) (A21124) or Alexa Fluor 594 Goat anti-chicken (A11042) and Alexa Fluor 488 Goat anti-rabbit IgG (A11008), all diluted 1:1000 (Life Technologies, Carlsbad, CA, USA). Cell nuclei were counterstained with Hoechst 33342 nucleic acid stain (H1399, Life Technologies).
5
Multicolor Flow Cytometry Antibody Panel
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Rabbit polyclonal antibody against calreticulin (PA3-900) was purchased from ThermoFisher (Rockford, IL, USA). Alexa Fluor 488 goat anti-rabbit IgG (A11008) was purchased from Life Technology (Eugene, OR, USA). PE-conjugated anti-mouse CD4 (clone RM4-5),PE/Cy7-conjugated anti-mouse CD8 (clone 53-6.7), FITC-conjugated anti-mouse CD25 (clone 3C7), Alexa Fluor 488-conjugated anti-mouseFoxP3 (clone 150D), APC-conjugated anti-mouse Gr-1 (clone RB6-8C5) and FITC-conjugated anti-mouse CD11b (clone M1/70) antibodies were purchased from BioLegend (San Diego, CA). PerCP-eFluor 710-conjugated anti-mouse CD3 (clone 17-A2)and APC eFluor 780-conjugatedanti-mouse CD127 (clone A7R34) antibodies were purchased from eBioscience (San Diego, CA).
6
Antibodies for Characterizing O-GlcNAc Network
7
Immunofluorescence Analysis of Caveolin-1
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Anti-clathrin heavy chain antibody (ab21679), anti-dynamin 2 antibody (ab3457) were obtained from Abcam. Caveolin-1 polyclonal antibody (PA5-17447), Alexa Fluor 488 goat anti-rabbit IgG (A11008) and horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody (31458) were from Thermo Fisher Scientific.
8
Measuring Lysosomal Size in BMDMs
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For assessing lysosomal size, we performed fluorescence confocal microscopy by staining for the lysosomal membrane marker lysosomal-associated membrane protein 1 (LAMP-1) followed by quantification of the number of lysosomes based on size. For this staining, fresh BMDM were fixed in paraformaldehyde (4%) and permeabilized in Triton-X (0.1%)/BSA (0.2%) solution. BMDM were incubated with the primary (1:100, rabbit polyclonal Lamp1, ab24170, Abcam, Cambridge, United Kingdom) and secondary (1:200, Alexa fluor 488 goat anti-rabbit IgG, A11008, Thermo Fisher Scientific, Waltham, Massachusetts, USA) antibodies and finally, sections were enclosed with glycerol mounting medium (DABCO-DAPI). Confocal pictures were taken with a LEICA DMI 4000 microscope (Leica microsystems, Wetzlar, Germany), providing 3D images of BMDM and their lysosomes.
9
Immunohistochemical Analysis of Microglial Activation
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Animals were perfused, and immunohistochemical procedures were performed as described later. Rapidly frozen sections with 20 μM were co-incubated with primary anti-AIF1 antibody (Wako Pure Chemical Industries, Chuo-ku, Osaka, Japan, 019-19741) and anti-LAMP2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA, sc-19991) overnight at 4°C. Secondary AlexaFluor 488 goat anti-rabbit IgG (A-11008) or AlexaFluor 594 goat anti-mouse (A-11032) from Thermo Fisher Scientific Waltham, MA, USA, was added for 2 h to detect Iba1 and LAMP2, followed by mounting of sections with prolong gold antifade reagent with 4,6-diamidino-2-phenylindole (Thermo Fisher Scientific, Waltham, MA, USA, P36935). Fluorescent images were acquired on a Zeiss Observer. AxioVs 40 4.8.0.0 software (Carl Zeiss, Thornwood, NY, USA) was used to process the images.
10
Immunofluorescence Assay for OCTN2
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Cells were seeded onto chamber slides at the density of 10,000 cells/well. When confluent, cells were fixed with 4% formaldehyde in PBS for 15 min at room temperature. Cells were subsequently permeabilized using 0.1% Triton X-100 in PBS for 15 min, and then incubated in blocking solution (PBS containing 0.1% Triton X-100, 3% goat serum, and 2% bovine serum albumin) for 30 min at room temperature, followed by overnight incubation at 4°C with anti-OCTN2 (LS-C681354, RRID:AB_2889180) diluted in PBS containing 0.1% Triton X-100 and 3% goat serum. Cells were washed with PBS, and then incubated for 1 h at room temperature with Alexa Fluor 488 goat anti-rabbit-IgG (A-11008, RRID:AB_143165, Thermo Fisher Scientific, Carlsbad, CA, United States) diluted in PBS-T. Cells were washed and mounted with Crystal Mount (GeneTex, Irvine, CA, United States) for picture acquisition using a Leica DMR upright fluorescence microscope (Leica Microsystems, Wetzler, Germany).
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