Immunofluorescence staining was performed as previously described (27 (link), 28 (link)). Briefly, intact islets were dispersed into single islet cells using TrypLE (Thermo Fisher Scientific, Waltham, MA, USA). Dispersed islets were then loaded onto the slides using a Shandon Single Cytofunnel (Thermo Fisher Scientific). The slides were fixed with 4% paraformaldehyde and incubated with primary anti-insulin (Agilent Technologies, Santa Clara, CA, USA), anti-glucagon (Abcam, Cambridge, MA, USA), and anti-Ki67 (Abcam) overnight at 4°C. Secondary anti-guinea pig Alexa488 (Thermo Fisher Scientific), anti-mouse Alexa555 (Abcam) and anti-rabbit Cy5 (Thermo Fisher Scientific) were used to detect the target proteins. Images were obtained using a Zeiss Axioscan Slide Scanner (Zen, Blue Edition, v.2.3.69.1000; Carl Zeiss GmbH). All image quantifications were carried out by HALO (Indica Labs, v.2.0.1145.14; Corrales, NM, USA). Cell proliferation rate was calculated by normalizing Ki67+ insulin+/glucagon+ cells to total insulin+/glucagon+ cells.
Anti rabbit cy5
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-rabbit Cy5 is a fluorescent dye-labeled secondary antibody used for the detection and visualization of rabbit primary antibodies in various immunodetection techniques, such as Western blotting, immunohistochemistry, and flow cytometry. The Cy5 dye, which emits light in the far-red/near-infrared range, provides a sensitive and specific signal for the labeling of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ventana discovery xt, by Roche (2 mentions)
Shandon single cytofunnel, by Thermo Fisher Scientific (1 mentions)
Axioscan slide scanner, by Zeiss (1 mentions)
Anti glucagon, by Abcam (1 mentions)
Anti insulin, by Agilent Technologies (1 mentions)
Anti ki67, by Abcam (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Anti cd45 clone hi30, by BioLegend (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Aperio versa 200, by Leica (1 mentions)
Anti cd3, by Agilent Technologies (1 mentions)
Blocking solution, by Agilent Technologies (1 mentions)
Anti geminin, by Proteintech (1 mentions)
Anti mouse cy3, by Thermo Fisher Scientific (1 mentions)
Multiimage 3, by Bio-Techne (1 mentions)
Rat anti brdu igg, by Abcam (1 mentions)
Alexa fluor 700 conjugated goat anti rabbit or goat anti mouse antibodies, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti neun, by Merck Group (1 mentions)
Mouse monoclonal anti nr2b, by Merck Group (1 mentions)
7 protocols using anti rabbit cy5
1
Immunofluorescence Staining for Islet Cell Analysis
2
Multiplexed Immunofluorescent Analysis of Tissue Samples
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TMA paraffin blocks were cut into 5 μm sections. Hematoxylin Eosin staining was used for visual evaluation of morphology to ensure comparable tissue samples were used for analysis. Multiplexed Immunofluorescent (mxIF) stain was performed with following antibodies: anti-PanCK, Clone AE1/AE3 (Invitrogen); anti-CD45, Clone HI30 (Biolegend); anti-CD3 (Agilent Inc., Dako); anti-HLADR, Clone SPM288 (Novus Biologicals LLC.). Multistep mxIF staining was perform, where after blocking, in a first step tissue was incubated with mouse anti-CD45 antibodies, followed by Fab fragment anti-mouse-Cy3 (Jackson ImmunoResearch). Tissue was washed well to remove unbound antibodies, blocked with mouse IgG and incubated with directly conjugated mouse PanCK-FITC, HLADR-Cy7 and rabbit anti-CD3 antibodies. Next, after washing, CD3 was detected in additional step with anti-rabbit-Cy5 (Thermo Fisher Scientific) antibodies. Nuclei were stained with DAPI (Thermo Fisher Scientific). Slides were coverslip with prolong gold (Invitrogen) and dried overnight. Whole slide imaging was performed on Aperio Versa 200 (Leica) scanner.
3
Immunohistochemical Analysis of GMNN
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Harvested tumors were fixed with formalin and embedded in paraffin. Tissue sections were cut with thick of four micrometers using a microtome. Antigen retrieval step and primary antibody staining were performed using the automated Ventana Discovery XT staining system (Ventana Medical Systems, Oro Valley, AZ, USA) according to the manufacturer’s instructions. Anti-GMNN antibodies were added on slides and incubated at 37 °C for 60 min. Then, blocking solution Dako (Agilent Technologies, Palo Alto, CA, USA) was incubated for 20 min followed by secondary antibodies step (anti-rabbit Cy5; Life Technologies Inc.) for 45 min. To quench tissue autofluorescence, slides were incubated for 15 min with Sudan Black (0.1% (w/v in 70% ethanol). Slides were then mounted with ProLong® Gold Antifade Mountant with DAPI and stored at 4 °C. To acquire images, a Zeiss microscopy was used and quantification was performed using ImageJ software.
4
Immunofluorescence Staining of Geminin in Tissue Sections
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Tumors were harvested, formalin-fixed, and paraffin-embedded. According to the manufacturer’s instructions, 4-μm-thick tissue sections were cut with a microtome and stained using the automated Ventana Discovery XT staining system (Ventana Medical Systems). Antigen retrieval was performed in Cell Conditioning 1 solution, and slides were incubated with anti-Geminin (Proteintech Group) antibodies in PBS at 37°C for 60 minutes. A negative control slide using PBS instead of the primary antibody was prepared in parallel. On the bench, slides were incubated for 20 minutes with blocking solution (Dako, Agilent Technologies) followed by washing with PBS and by incubation with the secondary antibody (anti-rabbit Cy5; Life Technologies Inc.) in PBS at room temperature for 45 minutes. Finally, after washing with PBS, slides were incubated for 15 minutes at room temperature with a 0.1% (w/v) solution of Sudan Black in 70% ethanol to quench tissue autofluorescence. Following a PBS wash, slides were mounted using ProLong Gold Antifade Mountant with DAPI and stored at 4°C. Images were taken with a Zeiss microscope and quantification was performed with ImageJ software.
5
Western Blot Quantification of Acot Proteins
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Tissue total lysates were prepared in lysis buffer (50mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1% Triton X-100) and 20–30 μg of protein were separated by SDS PAGE electrophoresis and transferred to nitrocellulose membranes. Acot1 (ab133948, Abcam, Cambridge, MA), Acot7 (affinity purified antibody [41 (link)]), and Hsc70 (sc-59570, Santa Cruz, Dallas, TX) coupled with anti-rabbit Cy5 (Invitrogen, Grand Island, NY) or anti-mouse Cy3 (Invitrogen) were visualized with Alpha Innotech MultiImage III and quantified using Alpha Innotech FluorChem Q Software (Santa Clara, CA), and data was normalized to Hsc70 expression.
6
Immunohistochemistry and Immunoblotting Assays
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Ro25-6981 hydrochloride hydrate was purchased from Sigma-Aldrich (Millipore Sigma, USA), and was administered 30 min (min) before behavioral tests. The following primary antibodies and dilutions were used for immunohistochemistry (IHC) staining: rat anti-BrdU IgG (1:1,000; Abcam), mouse monoclonal anti-GFAP (1:1,000; Millipore), mouse monoclonal anti-NeuN (1:200; Millipore), anti-rat FITC (1:200; Invitrogen, Carlsbad, CA), anti-mouse Rhodamine Red-X (1:200; Invitrogen), and anti-rabbit Cy5 (1:200; Invitrogen). The antibodies below were used for immunoblotting: mouse monoclonal anti-NR2B (1:1,000; Millipore), mouse monoclonal anti-BDNF (1:1,000; Millipore), rabbit monoclonal anti-phospho CREB and anti-CREB (both 1:1,000; Millipore) and β-actin (1:10,000; Abcam). Alexa Fluor 700 conjugated goat anti-rabbit or goat anti-mouse antibodies (both 1:20,000; Invitrogen, Eugene, OR) were used as secondary antibodies.
7
Immunofluorescence and Telomere FISH Analysis
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Cells were grown on coverslips, fixed with 4% PFA, followed by 2 washes with cold PBS, permeabilized with 0.25% Triton X-100 in PBS, 3× washed with PBS, and blocked with 1% BSA in PBST at room temperature. Coverslips were then incubated with antibody solution in 1% BSA PBST. Antibodies were anti-phospho-H2AX (Ser139) (Millipore; 07-164), anti-53BP1 (Abcam; ab36823), anti-p21 (F-5) (Santa Cruz; sc-6246), anti-BLM (Sigma-Aldrich; HPA005689) and anti-Phospho RPA32 (S33) (Bethyl; A300-246A). Cells were incubated with antibody solution over night at 4°C followed by 3 washes in PBS, 1 h incubation with an appropriate secondary antibody labeled with fluorophore, anti-mouse Cy3 (Sigma, C2181), anti-rabbit Cy3 (Jackson ImmunoResearch, 111-165-006) and anti-rabbit Cy5 (Invitrogen, 81-6116) and 3 washes in PBS. Finally, samples were mounted with DAPI fluorescence mounting medium and analyzed under a fluorescence microscope. For IF-FISH, immunofluorescence stainings were done as described above, and slides were shortly fixed in 2% PFA at room temperature. And then, slides were processed for telomere qFISH as described before.
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