Tissue preparation and analyses have been described in detail previously [16 (link)]. Pancreas tissue was collected 1 day after the final administration of mSTZ to analyse cleaved caspase-3 and at 9 weeks of age to analyse beta cell mass. For analysis of cleaved caspase-3, sections were treated in citrate buffer-based Target Retrieval Solution (Dako, Glostrup, Denmark) before primary antibody incubation. Sections were incubated overnight with primary antibodies to cleaved caspase-3 (1:300; #9661; Cell Signaling Technology, Danvers, MA, USA) and/or insulin (1:150; ab7842; Abcam, Cambridge, MA, USA). Secondary antibodies (Alexa fluor 488, 594; 1:500; A-11073, A-11037 or A-11076; Molecular Probes, Eugene, OR, USA) were applied and incubated for 90 min at room temperature. Antibodies used are commercially available and widely used for immunohistochemistry. Fluorescent images were taken using BZ-9000 Fluorescence Microscope (Keyence, Osaka, Japan). The number of islets used to analyse cleaved caspase-3 was 355. The total areas of islets, insulin-positive cells (beta cells) and cleaved caspase-3 were analysed using BZ-X analyser software (Keyence).
A 11076
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A-11076 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a versatile instrument designed for use in various scientific applications. The core function of the A-11076 is to perform precise measurements and analyses, but without further details, a more specific description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
G2654, by Merck Group (3 mentions)
A0564, by Agilent Technologies (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
A11073, by Thermo Fisher Scientific (2 mentions)
Citrate buffer, by Merck Group (2 mentions)
A11001, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
A21202, by Thermo Fisher Scientific (2 mentions)
A11029, by Thermo Fisher Scientific (2 mentions)
Ab7842, by Abcam (1 mentions)
A11037, by Thermo Fisher Scientific (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Bz x analyser software, by Keyence (1 mentions)
Bz 9000 fluorescence microscope, by Keyence (1 mentions)
Microscope slides, by Agar Scientific (1 mentions)
Hoechst, by Merck Group (1 mentions)
A11012, by Thermo Fisher Scientific (1 mentions)
Agc 001, by Alomone (1 mentions)
Ab5622, by Merck Group (1 mentions)
Is20015, by Immunological Sciences (1 mentions)
14 protocols using a 11076
1
Pancreas Tissue Analyses for Cleaved Caspase-3 and Beta Cell Mass
2
Immunohistochemical Labeling of Synaptic Proteins
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Coronal sections (50 µm) were first permeabilised overnight with 0.25% Triton X-100 in 1 × phosphate-buffered saline (PBST, pH 7.4) at 4 °C. After 3 washes with 1× PBST, non-specific binding was blocked by incubating the sections with 10% normal goat serum (NGS) in 1× PBST for 1 h at RT. The sections were immunolabelled for GluN1 (1:500; AGC-001, Alomone Labs), Shank2 (1:500; 162 204, Synaptic Systems) or Shank3 (1:500; 162 302, Synaptic Systems) and synapsin1/2 (1:500; 106 004, Synaptic Systems) with primary antibody solution prepared in 1× PBST containing 1% NGS for 72 h at 4 °C. The sections were washed in 1× PBST, incubated for 4 h at RT with secondary antibodies (goat anti‐guinea pig IgG‐Alexa Fluor 594, 1:500; A11076, Molecular Probes; goat anti-rabbit IgG-Alexa Fluor 594, 1:500; A11012, Molecular Probes; goat anti‐rabbit IgG‐Alexa Fluor 647, 1:500; Molecular Probes, A21245). No primary antibody controls showed no immunostaining signal for all antibodies used. The sections were further washed in 1× PBST and incubated with Hoechst (Sigma) for 30 min at RT and mounted on microscope slides (Menzel Glaser) in Citifluor mounting medium (Agar Scientific, AF1).
3
Differentiation of NPCs to Glutamatergic Neurons
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A total of 4 × 104 NPCs were seeded on Matrigel-coated covers and differentiated to glutamatergic neurons. Cells were fixed in 4% paraformaldehyde and processed as previously described [18 (link)]. Immunofluorescence was performed using specific antibodies: rabbit anti microtubule-associated protein 2, Map2 (1:400; AB5622, Sigma-Aldrich); guinea pig anti vesicular glutamate transporter 1, Vglut1 (1:200; 135304, Synaptic System, Goettingen, Germany); Alexa fluor 488 donkey anti rabbit IgG (1:800; IS20015, Immunological Sciences, Roma, Italy); Alexa fluor 594 goat anti guinea pig IgG (1:800; A11076, ThermoFisher Scientific, Monza, Italy). Images were acquired by fluorescence microscope Zeiss Axio Observer.Z1 equipped with Hamamatsu EM-CCD 9100-02 camera and Volocity acquisition software.
4
Immunofluorescence Imaging of Pancreatic Cells
5
Multicolor Immunostaining of Isolated Islets
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Immunostaining was conducted on isolated islets, fixed in 4% paraformaldehyde before being permeabilised using 0.1% Triton X-100 (Sigma). After blocking with 5% goat serum, islets were incubated overnight (4°C) with primary antibodies before incubation with secondary antibodies. Fluorescent staining was visualised using a laser-scanning confocal microscopy (BioRad) controlled by LaserSharp2000 (BioRad).
Antibodies used in this study were: rabbit FITC 495-conjugated anti-GFP (1:250; DS-PB-00926; InsightBio, Wembley, United Kingdom), mouse anti-glucagon (1:1000; G2654; Sigma), guinea-pig anti-insulin (1:400; A0564; Dako, Santa Clara, California, United States), goat anti-somatostatin (1:100; sc-7819; Santa Cruz Biotechnology, Dallas, Texas, United States); Alexa Fluor 633 goat anti-mouse IgG (1:500; A-21052; ThermoFisher); Alexa Fluor 594 goat anti-guinea pig IgG (1:500; A-11076; ThermoFisher); and Alexa Fluor 546 donkey anti-goat IgG (1:100; A-11056; ThermoFisher).
Antibodies used in this study were: rabbit FITC 495-conjugated anti-GFP (1:250; DS-PB-00926; InsightBio, Wembley, United Kingdom), mouse anti-glucagon (1:1000; G2654; Sigma), guinea-pig anti-insulin (1:400; A0564; Dako, Santa Clara, California, United States), goat anti-somatostatin (1:100; sc-7819; Santa Cruz Biotechnology, Dallas, Texas, United States); Alexa Fluor 633 goat anti-mouse IgG (1:500; A-21052; ThermoFisher); Alexa Fluor 594 goat anti-guinea pig IgG (1:500; A-11076; ThermoFisher); and Alexa Fluor 546 donkey anti-goat IgG (1:100; A-11056; ThermoFisher).
6
Immunofluorescence Staining of Frozen Tissue
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Frozen sections were washed in PBS with 0.2% Triton X-100 (PBST) and blocked with 5% normal donkey serum (NDS) for 1 h. The slides were then incubated with primary antibodies mixed in 1% NDS overnight at 4°C. Antibodies against TCF7L2 (1:500; 2569, Cell Signaling Technology), Cav3.1 (1:500; MABN464, Sigma-Aldrich, NeuroMab clone N178A/9), β-galactosidase (1:100; AB986, Merck Millipore), L1CAM (1:500; MAB5272, Merck Millipore), PAX6 (1:100; PRB-278P, BioLegend), KI-67 (1:100; AB9260, Merck Millipore), TUJ1 (1:65; MAB1637, Merck Millipore), NKX2-2 (1:50; 74.5A5, Developmental Studies Hybridoma Bank), SIX3 (1:100; 200-201-A26S, Rockland), POU4F1 (1:300; Fedtsova and Turner, 1995 (link)) were used. Sections were then incubated for 1 h with appropriate secondary antibody conjugated with Alexa Fluor 488 or 594 (1:500; A-21202, A-21207 and A-11076, Thermo Fisher Scientific). The slides were additionally stained with Hoechst 33342 (1:10,000; 62249, Thermo Fisher Scientific), washed and mounted with Vectashield Antifade Mounting Medium (H1000, Vector Laboratories).
7
Immunofluorescence Imaging of Pancreatic Cells
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IF was performed as described previously 82 (link),83 (link). Briefly, after antigen retrieval using citrate buffer (Sigma), 5μm-thick formalin-fixed pancreatic sections were incubated with anti-insulin (1:1000, Agilent Cat#A0564, RRID:AB_10013624) or anti-glucagon primary antibodies (1:1000, Sigma-Aldrich Cat#G2654, RRID:AB_259852) and AlexaFluor-594-conjugated goat anti guinea-pig (1:500, Molecular Probes Cat#A-11076, RRID:AB_141930) and AlexaFluor-488-conjugated goat anti-mouse antibodies (1:500, Thermo Fisher Scientific Cat#A-11001, RRID:AB_2534069). Images were processed for morphometry using ImageJ software by an observer blind to experimental groups.
8
Immunofluorescence Analysis of Mouse Brain Sections
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Immuno uorescence staining was performed on frozen coronal cerebral sections of mice. Mice were xed with 4% paraformaldehyde after deep anesthesia. And mouse brains were post-xed for another 6 ~ 8 h. After xation and dehydration in sucrose, the brains were then cut into 12-μm-thick sections. The brain sections were washed with PBS and then incubated with the following primary antibodies overnight at 4 ºC in a humidi ed atmosphere: rabbit anti-BDNF (1:100, ab108319, Abcam), guinea pig anti-NeuN (1:300, 266004, Synapse System, Gottingwn, Germany), rabbit anti-DCX (1:300, E6O6A, Cell Signaling Technology, Massachusetts, USA), chicken anti-GFAP (1:400, GTX85454, Gentex, Texas, USA), rabbit antip(Ser727)STAT3 (1:200, ab267373, Abcam) and mouse anti-p(Tyr705)STAT3 (1:200, M9C6, Cell Signaling Technology). Then, the samples were incubated with Alexa 488-conjugated goat anti-guinea pig (1:300, A11076, Thermo Fisher, Waltham, USA), Alexa 594-conjugated donkey anti-rabbit (1:300, ab150064, Abcam), and Alexa 488-conjugated donkey anti-rabbit (1:300, ab150073, Abcam) antibodies for 2 h in a dark environment at room temperature. Finally, the sections were viewed and images were acquired using an Olympus (Bx51, Japan) uorescence microscope.
9
Immunostaining of Drosophila Wing Discs
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Wing discs were dissected form 3rd instar larvae in Schneider’s medium (Gibco by Life Technologies) and fixed for 20 min in 4% PFA in PBS. Permeabilization was allowed to proceed for 30 min in 0.2% Triton X-100 in PBS (PTx), followed by 60 min blocking in 0.2% BSA and overnight incubation with the primary antibody at 4°C. After washing, the secondary antibody incubation was performed for 60 min at RT, and washed again in PTx and finally with PBS.
Extracellular Wg staining was performed as described previously (Strigini and Cohen, 2000 (link)). Wing discs were mounted in Vectashield Mounting Medium (Vector Laboratories). Images were acquired at a Leica TCS Sp5 confocal microscope. Signal intensities were assessed using FIJI. Separate channel images were assembled using Adobe Photoshop CS6.
The following antibodies were used: Mouse Anti-Wg (4D4s, obtained from Developmental System Hybridoma Bank) 1:5 for extracellular and 1:50 for total staining, Rabbit-anti-Dll 1:200 (a gift from S. Carroll), Guinea Pig anti-Sens 1:300 (Gross et al., 2012 (link)). Secondary antibodies used were anti-guinea pig-Alexa594 (1:500 (A11076), Invitrogen), anti-mouse-Alexa594 (1:500, (A11005), Invitrogen).
Extracellular Wg staining was performed as described previously (Strigini and Cohen, 2000 (link)). Wing discs were mounted in Vectashield Mounting Medium (Vector Laboratories). Images were acquired at a Leica TCS Sp5 confocal microscope. Signal intensities were assessed using FIJI. Separate channel images were assembled using Adobe Photoshop CS6.
The following antibodies were used: Mouse Anti-Wg (4D4s, obtained from Developmental System Hybridoma Bank) 1:5 for extracellular and 1:50 for total staining, Rabbit-anti-Dll 1:200 (a gift from S. Carroll), Guinea Pig anti-Sens 1:300 (Gross et al., 2012 (link)). Secondary antibodies used were anti-guinea pig-Alexa594 (1:500 (A11076), Invitrogen), anti-mouse-Alexa594 (1:500, (A11005), Invitrogen).
10
Immunofluorescence Labeling with Fluorescent Secondary Antibodies
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The following secondary antibodies (dilution, antibody, and vendor) were used: Alexa Fluor 488 goat anti–rabbit IgG (H+L) (1:500, A11008, Invitrogen); Alexa Fluor 495 Fab’2 fragment of goat anti–mouse IgG (H+L) (1:500, A11020, Invitrogen); Alexa Fluor 488 goat anti–mouse IgG (H+L) (1:500, A11029, Invitrogen); Alexa Fluor 594 goat anti–guinea pig IgG (H+L) (1:500, A11076, Invitrogen); goat anti–mouse IgG (H+L)-HRP conjugate (1:2000, Bio-Rad 170-6516); Alexa Fluor 488 goat anti–rat IgG (H+L) (1:500, A11006, Invitrogen); and goat anti–rabbit IgG (H+L)-HRP conjugate (1:2000, Bio-Rad 170-6515).
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