In all, 10- to 12-week-old mice (n = 7–8 per group) were perfused transcardially with 4% paraformaldehyde. The brains were then frozen, sectioned at 30-µm thick, and processed for immunofluorescence using standard procedures7 (link). The primary antibodies used for IHC were as follows: sheep anti-αMSH antibody (1:40,000, Millipore, cat #AB5087), rabbit anti-AgRP (1:1,000, Phoenix Pharmaceuticals, cat #H003-53), rabbit anti-LC3B (1:500, Cell Signaling, cat #2775), rabbit anti-p62 (1:1,000, Abcam, cat #ab64134), rabbit anti-GFP (1:1,000, Invitrogen, cat #A-6455), guinea pig anti-insulin (1:500, Abcam, cat #ab7842), and rabbit anti-vesicular acetylcholine transporter (VAChT, 1:500, Synaptic Systems, cat #139103). The primary antibodies were visualized with Alexa Fluor 568 donkey anti-sheep IgG (1:200, Invitrogen, cat #A21099), Alexa Fluor 488 donkey anti-rabbit IgG (1:200, Invitrogen, cat #A21206), Alexa Fluor 488 donkey anti-mouse IgG (1:200, Invitrogen, cat #A21202), Alexa Fluor 568 donkey anti-rabbit IgG (1:200, Invitrogen, cat #A10042), or Alexa Fluor 488 goat anti-guinea pig IgG (1:200, Invitrogen, cat #A11073). The sections were counterstained using bis-benzamide (1:10,000, Invitrogen, cat #H3569) to visualize cell nuclei.
Alexa fluor 488 goat anti guinea pig igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Alexa Fluor 488 goat anti-guinea pig IgG is a fluorescently labeled secondary antibody used to detect and visualize guinea pig immunoglobulin G (IgG) in various immunoassay applications. It is conjugated to the Alexa Fluor 488 dye, which provides bright green fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 568 donkey anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Goat anti chicken igy alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Guinea pig anti insulin, by Agilent Technologies (2 mentions)
Axiovert 200m microscope, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Gp62 c, by Progen Biotechnik (2 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Anti mrgprc11, by Proteintech (2 mentions)
Alexa fluor 555 donkey anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 donkey anti rat igg, by Thermo Fisher Scientific (2 mentions)
Ib4 alexa 488, by Thermo Fisher Scientific (2 mentions)
Mab356, by Fortrea (2 mentions)
Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Rabbit anti lc3b, by Cell Signaling Technology (1 mentions)
Sheep anti αmsh antibody, by Merck Group (1 mentions)
Rabbit anti agrp, by Phoenix Pharmaceuticals (1 mentions)
Vacht, by Synaptic Systems (1 mentions)
Alexa fluor 568 donkey anti sheep igg, by Thermo Fisher Scientific (1 mentions)
Bis benzamide, by Thermo Fisher Scientific (1 mentions)
19 protocols using alexa fluor 488 goat anti guinea pig igg
1
Immunofluorescence Analysis of Mouse Brain
2
Quantifying Pancreatic Cell Mass Using Immunofluorescence
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Permeabilized slices were prepared as described previously (20 (link)) and blotted with primary antibodies, anti-guinea pig insulin (1:200; Dako, Ely, UK), anti-rabbit glucagon (GCG; 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-rabbit NPTX2 (5 μg/ml; Abcam, Cambridge, UK). Slices were visualized with Alexa Fluor 488 goat anti-guinea pig IgG and with Alexa Fluor 568 donkey anti-rabbit IgG (Invitrogen, Paisley, UK) using an Axiovert 200 M microscope (Zeiss, Welwyn Garden City, UK). ImageJ software (Wayne Rasband, NIH, Bethesda, MD, USA) was used to calculate β- and α-cell mass. OPT of whole pancreata stained with anti-insulin antibodies was performed as described previously (20 (link)).
3
Immunofluorescence Analysis of Cytoskeletal Proteins
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Primary antibodies: Anti-MAP1B HC rabbit pAb (BR18, provided by Beat M. Riederer, Department of Cell Biology and Morphology, University of Lausanne, Switzerland), anti-Nephrin guinea pig pAb, GP-N2 (Progen, Heidelberg, Germany), anti–α-tubulin mouse mAb, DM1A (Sigma, Saint Louis, USA). Nuclear staining reagents and fluorophore-conjugated secondary antibodies were obtained from Invitrogen: Hoechst 33258; Alexa Fluor 488 goat anti-guinea pig IgG (A11073); Alexa Fluor 546 donkey anti-rabbit (A10040), Alexa Fluor 488 donkey anti-mouse IgG (A-21202).
4
Immunofluorescent Staining of Kidney and Pancreas
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Immunofluorescent staining of kidney and pancreas was performed as previously described [18] (link). Briefly, renal expression of type IV collagen was detected a rabbit antibody for type IV collagen (Millipore, Temecula, CA, USA) followed by Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA). Similarly, pancreatic β-cells were detected using guinea pig anti-insulin (Abcam, Cambridge, UK) followed by Alexa Fluor 488 goat anti-guinea pig IgG (Invitrogen). The positive area of type IV collagen in the glomerulus was calculated in the same way as MMI. The proportion of β-cells in the pancreatic tissue was calculated using BIOZERO software (Keyence). The insulin-positive area relative to the area of the whole pancreatic tissue was analyzed in more than 100 islets per group.
5
Quantifying Pancreatic Cell Populations
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Isolated pancreata were fixed in 10% (v/v) formalin for 24 hours and embedded in paraffin wax. Sections (5 μm) were cut and fixed on Superfrost slides. Slides were permeabilized as detailed in Ref. 38 (link) and blotted with the following primary antibodies: antiguinea pig insulin (1:200; Dako) and antirabbit glucagon (1:100; Santa Cruz Biotechnology). Slides were visualized using an Axiovert 200M microscope (Zeiss) with Alexa Fluor 488 goat antiguinea pig IgG and with Alexa Fluor 568 donkey antirabbit IgG (Invitrogen). For examination of apoptosis, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays were performed on the above sections using a DeadEnd Fluorometric TUNEL system kit (Promega) according to the manufacturer's instructions. ImageJ software (Wayne Rasband, National Institute of Mental Health) was used to calculate the mean intensity of insulin staining in the insulin-positive area, β- and α-cell mass, and the number of TUNEL-positive cells of all visible islets. For β-cell mass estimation by immunocytochemistry, we determined the percentage of pancreatic surface that was insulin positive, as measured in whole pancreas sections separated by 25 μm in the z-axis.
6
Quantifying Dendritic Development of DCX-Positive Cells
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Six rats were measured in each group. Using the free-floating method (Wang et al., 2015), brain sections were washed three times with 0.01 M PBS for 10 minutes each, blocked with 5% goat serum (Bioss Biotechnology, Beijing, China) for 90 minutes, incubated with Guinea pig anti-DCX polyclonal antibody (1:800; Millipore, Billerica, MA, USA) at 4°C overnight, and then washed three times with 0.01 M PBS for 10 minutes each. All samples were incubated with Alexa Fluor 488 goat anti-guinea pig IgG (1:800; Invitrogen, Carlsbad, CA, USA) at room temperature for 2 hours in the dark, and washed three times with 0.01 M PBS for 10 minutes each. Afterwards, all sections were mounted with anti-fluorescence quenching mounting medium. Five sections through the dentate gyrus of each rat were quantified with NIH ImageJ software (Kralic et al., 2005). The dendritic development of doublecortin (DCX)-positive cells in the hippocampal dentate gyrus was observed with a laser scanning confocal microscope (FV1000; Olympus, Tokyo, Japan). The three-dimensional structure of dendrites was reconstructed after a series of layers of DCX-positive cells in the Z-axis direction were magnified 200-fold under the confocal microscope. Dendrites and their branches were measured and described with NIH ImageJ software (Stumm and Höllt, 2007).
7
Fluorescence-based Assay for VZV Antibodies
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FAMA assay was performed with slight modification from previously described methods [29 (link)]. Briefly, MRC-5 cells were grown to confluency in 175 T flasks and infected with VZV isolates at 0.003 m.o.i. When cytopathic effects were observed in ~ 50- 60% of the cells, the cells were washed three times with PBS. Infected cells were detached from the flasks and incubated with serially-diluted sera at room temperature for 30 min. After washing with PBS, cell preparations were incubated with Alexa Fluor®488 goat anti-guinea pig IgG (Invitrogen) and mounted on slides. After overlaying with mounting media containing DAPI, the slide was covered with a cover-glass and observed under a fluorescence microscope (Nikon). Titers were defined as the reciprocal of the highest dilution causing bright fluorescent ring around the surface of cells. Titers of ≥ 1:4 were considered positive. Pooled sera from MAV/06 immunized guinea pigs were pre-treated with MRC-5 cells before incubation with virus-infected cells in order to remove immune responses to cellular debris within vaccine ingredients. Non-specific reactions to mock-infected cells were confirmed using both vaccine groups.
8
Immunofluorescence Imaging of SQSTM1
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MM cells were seeded on poly-l-lysine-coated slides, fixed with 4% paraformaldehyde, and permeabilized with PBS 0.1% Triton X-100. Cells were stained with guinea pig SQSTM1 antisera (1:200), C-terminal (Progen, GP62-C), rinsed in PBS, and stained with Alexa Fluor 488 goat anti-guinea pig IgG (1:500: Life Technologies, A11073) and Hoechst 33,342 (Life Technologies, H3570). Images were acquired with Deltavision (Applied Precision, Issaquah, WA) with an Olympus 60× objective with a numerical aperture of 1.42. For every sample several stacks were acquired on the z-axis. Images were deconvoluted using SoftWorks 3.5.0 (Applied Precision) and processed with ImageJ. More than 80 cells per sample were counted and analyzed.
9
Immunohistochemical Analysis of 11β-HSD1 in Murine Pancreas
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Paraffin sections of the pancreas taken from 3–5 month old, male MT-IGF and wild-type littermates were dewaxed, rehydrated, and blocked with 10% donkey serum, followed by incubation with rabbit anti-11β-HSD1 antibodies (1:100; H-10 sc-20175, Santa Cruz and ab83522, Abcam, Cambridge, MA) at 4°C overnight. After washing with PBS, sections were independently stained with anti-glucagon (C-18 sc-7779, Santa Cruz) and guinea pig polyclonal anti-insulin (ab7842, Abcam) followed by Alexa Fluor 594 conjugated donkey anti-rabbit IgG (H+L) and Alexa Fluor 488 goat anti-guinea pig IgG (Life technologies, Carlsbad, CA) [14 (link), 15 (link)]. The images were analyzed using Axioshop 2 plus microscope (Carl Zeiss, Jena, Germany), Retiga 1300 digital camera, and Northern Eclipse software (Empix Imaging, Mississauga, ON). Paraffin sections of liver and pancreata taken from 3–5 month old male wild-type mice were incubated overnight with anti-11β-HSD1 (ab83522) with or without a specific blocking peptide (ab99223) at 4°C. After washing, the sections were incubated with secondary antibody and stained with diaminobenzidine substrate (Vector Laboratories). The microscopic images were analyzed using BX61 UIS2 Optical System microscope (Olympus) and Olympus stream software.
10
Immunofluorescence Staining of SQSTM1
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HEp-2 cells were seeded on slides, fixed with 4% paraformaldehyde, and permeabilized with PBS 0.1% Triton X-100. Cells were stained with guinea pig SQSTM1 antisera (1:200), C-terminal (Progen, GP62-C, Brisbane, Australia), rinsed in PBS, and stained with Alexa Fluor 488 goat anti-guinea pig IgG (1:500, Life Technologies, Carlsbad, CA) and Hoechst 33,342 (Life Technologies, Carlsbad, CA). Images were acquired with Leica TCS SP8 confocal microscope with a 63× objective (oil) with a numerical aperture of 1.4.
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