Milk gland tubules and fat body were removed from tsetse fly females at six-seven days after the previous birth. The combined milk gland/fat body was placed into Carnoy’s fixative for a five-six day fixation period.4 (link),72 (link) Antisense/sense digoxigenin-labeled RNA probes for cct1 were generated using the MAXIscript T7 transcription kit following manufacturer’s protocol (Ambion, Austin, TX, USA) using a primer set with a T7 primer (Table S1 ). Antibody solutions were made using α-Digoxigenin-rhodamine, Fab fragments (Roche Applied Science, Penzberg, Germany) for FISH probe detection (1:200 dilution) and rabbit α-GmmMGP (1:2500) antibodies. Alexa Fluor 488 goat α-rabbit IgG (Invitrogen) at a dilution of 1:500 was added as a secondary antibody for immunohistochemistry.4 (link),72 (link) Slides were mounted in VECTASHIELD Mounting Medium with DAPI (Vector laboratories Inc. Burlingame, CA, USA). Samples were observed using a Zeiss Axioskop2 microscope (Zeiss, Thornwood, NY, USA) equipped with a fluorescent filter and viewed and imaged at 400× magnification. Images were captured using an Infinity1 USB 2.0 camera and software (Lumenera Corporation, Ottawa, ON, Canada) and merged in Adobe Photoshop.
Alexa fluor 488 goat α rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 goat α-rabbit IgG is a secondary antibody conjugate that binds to rabbit immunoglobulin G (IgG) and is labeled with the Alexa Fluor 488 fluorescent dye. It is designed for use in immunofluorescence, flow cytometry, and other applications requiring the detection of rabbit primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Maxiscript t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Axioskop 2 microscope, by Zeiss (2 mentions)
Anti smad5, by Proteintech (1 mentions)
Anti myc tag, by Cell Signaling Technology (1 mentions)
Epics xl mcl, by Beckman Coulter (1 mentions)
Anti smad1, by Cell Signaling Technology (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Alexa fluor 488 goat α mouse igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
α pdia1, by Proteintech (1 mentions)
α proinsulin, by HyTest (1 mentions)
Streptavidin alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
αkeratin 5, by Fortrea (1 mentions)
Lsm 710 spectral, by Zeiss (1 mentions)
5 protocols using alexa fluor 488 goat α rabbit igg
1
Visualization of Tsetse Fly Milk Gland and Fat Body
2
Localizing Milk Gland Proteins in Postpartum Tissue
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Milk gland tubules intertwined with fat body were collected from mothers 0–2 hours after parturition. The combined milk gland/fat body was placed into Carnoy’s fixative for a five-six day fixation period [36 (link)]. Antisense/sense digoxigenin-labeled RNA probes were generated using the MAXIscript T7 transcription kit following manufacturer's protocol (Ambion, Austin, TX) using a primer set with a T7 primer (S1 Table ) [36 (link)]. Antibody solutions were made using α -Digoxigenin-rhodamine Fab fragments (Roche) for FISH probe detection (1:200 dilution) and rabbit α -GmmMGP (1:2500) antibodies [21 (link), 36 (link)]. Alexa Fluor 488 goat α -rabbit IgG (Invitrogen) at a dilution of 1:500 was added as a secondary antibody for immunohistochemistry [36 (link)]. Slides were mounted in VECTASHIELD Mounting Medium with DAPI (Vector laboratories Inc. Burlingame, CA). Samples were observed using a Zeiss Axioskop2 microscope (Zeiss, Thornwood, NY) equipped with a fluorescent filter and viewed and imaged at 400x magnification. Images were captured using an Infinity1 USB 2.0 camera and software (Lumenera Corporation, Ottawa, Ontario, Canada) and merged in Adobe Photoshop.
3
BMP2 Signaling in Myoblast Differentiation
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C2C12 cells were transfected as described. Where indicated, cells were starved in serum-free DMEM for 3 hours and stimulated with 10 nM BMP2 24 h post-transfection. Cells were harvested and fixed/permeabilised by incubation in ice cold 100% EtOH overnight at −20°C. After blocking in 1% BSA/1x PBS cells were co-stained with anti-myc tag (Cell Signaling), anti-Smad1 (Cell Signaling) or anti-Smad5 (Proteintech) antibodies recognized by R-phycoerythrin goat-α-mouse (#P852, Invitrogen) and Alexa Fluor®488 goat-α-rabbit IgG (#A11034, Invitrogen) antibodies. Immortalised human myoblasts were differentiated at confluence using OptiMEM containing 2% HS in the absence or presence of 10 nM BMP2. Cells were harvested and fixed/permeabilised using EtOH. Cells were stained using an α-IRS4 antibody (Origene) recognised by Alexa Fluor®488 goat-α-rabbit IgG antibody (#A11034, Invitrogen); as control nonspecific isotype control IgG (#3900, Cell Signaling) was used. Measurements were performed using an Epics XL-MCL flow cytometer (Beckman-Coulter). Data were evaluated using FCS3.0 Express (Denovo Software).
4
Immunohistochemical Analysis of Pancreatic Islets
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Pancreata were harvested and fixed in 4% PFA. Paraffin embedding, sectioning, and slide preparations were done in the SBP Histopathology Core Facility. Sections were stained with the following antibodies; α-glucagon (Abcam, K79bB10), α-PDIA1 (Proteintech, 11245–1-AP), α-proinsulin (HyTest Ltd., 2PR8, CCI-17), and DAPI (Fisher Scientific). Guinea pig polyclonal α-insulin antibody was produced in-house. For secondary antibodies, Alexa Fluor 488 goat α-rabbit IgG, Alexa Fluor 488 goat α-mouse IgG, Alexa Fluor 594 goat α-mouse IgG, and Alexa Fluor 594 goat α-guinea pig IgG antibodies were used (Invitrogen). Images were taken by Zeiss LSM 710 confocal microscope with a 40X objective lens. Scale bar, 20 µm.
For ß cell area measurement, pancreata were harvested, fixed in 4% PFA and embedded in paraffin. Three sections were prepared at 200 µm intervals for each pancreas and stained with guinea pig polyclonal insulin antibody and DAPI. Alexa Fluor 594 goat α-guinea pig IgG was used as a secondary antibody. Images were taken by an Aperio FL Scanner (Leica). Insulin stained ß cell area, islet area, and pancreas area were measured by Aperio Imagescope software.
For ß cell area measurement, pancreata were harvested, fixed in 4% PFA and embedded in paraffin. Three sections were prepared at 200 µm intervals for each pancreas and stained with guinea pig polyclonal insulin antibody and DAPI. Alexa Fluor 594 goat α-guinea pig IgG was used as a secondary antibody. Images were taken by an Aperio FL Scanner (Leica). Insulin stained ß cell area, islet area, and pancreas area were measured by Aperio Imagescope software.
5
Quantifying Myeloid Cell Subsets in Thymus
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