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Alexa fluor 594 conjugated affinipure donkey anti rabbit igg

Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom

Alexa Fluor 594-conjugated AffiniPure Donkey Anti-Rabbit IgG is a secondary antibody reagent that specifically binds to rabbit immunoglobulin G (IgG) antibodies. The antibody is conjugated with the Alexa Fluor 594 fluorescent dye, which can be detected using appropriate instrumentation.

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6 protocols using alexa fluor 594 conjugated affinipure donkey anti rabbit igg

1

Immunofluorescence Analysis of Lung Histology

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Lungs were prepared for histology and immunofluorescence as previously described (57 (link), 58 (link)). Frozen sections (8 μM) were fixed, permeabilized with 0.2% Triton-X, and blocked with 10% donkey serum and 3% bovine serum albumin (blocking buffer). Sections were then incubated overnight at 4°C in a humidified chamber with the following primary Abs: α-F4/80 (1:100, catalog 6640) and α–LOX-1 (1:100, catalog 60178) (Abcam). Sections were then washed and incubated with the following secondary Abs: Alexa Fluor 488–conjugated AffiniPure donkey anti-rat IgG (1:1,000, catalog 712-545-150) and Alexa Fluor 594–conjugated AffiniPure donkey anti-rabbit IgG (1:1,000, catalog 711-585-152) (Jackson Immunoresearch) at room temperature for 1 hour in a dark, humidified chamber. Secondary-only controls for LOX-1 (no α–LOX-1) and F4/80 (no α-F4/80) were also generated. Slides were then washed and counterstained with DAPI (Life Technologies) and mounted with FluorSave (MilliporeSigma). Visualization of histology and immunofluorescence was performed on a Leica DM4 LED light microscope equipped with a Leica DFC 7000T camera. Images were taken on dry, coverslipped slides using a 40× objective (total magnification = 400×) at room temperature with a numerical aperture of 0.8 using the Leica Application Suite X software. Images were processed using ImageJ 2.0.0-rc-69 (NIH).
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2

Immunofluorescence of Jejunum Tissue

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Immunofluorescence microscopy of the jejunum was performed as previously described with minor modifications.11 (link) For immunohistochemistry, tissues were fixed in 4% paraformaldehyde/PBS for 1 hour 20 minutes at 4°C prior to dehydration overnight in 20% sucrose/PBS at 4°C. The tissues were cut and placed into Tissue-Tek Crymold (Sakura Finetek, Torrence, CA, USA) containing one part Tissue-Tek OCT Compound (Sakura Finetek) and one part 20% sucrose/PBS prior to flash freezing with liquid nitrogen. Embedded molds were sectioned with a cryostat at 8 μm thickness onto glass slides and stained with primary antibodies for SRF (1:100, Cat No: sc-13029; Santa Cruz Biotechnology, Dallas, TX, USA), Ki67 (1:100, Cat No: RM-9106-S0; Thermo Scientific, Fremont, CA, USA) overnight at 4°C followed by staining with the secondary antibody Alexa Fluor 594-Conjugated AffiniPure Donkey Anti-Rabbit IgG (1:500, Cat No: 711-585-152; Jackson Immuno Research, West Grove, PA, USA) for 1 hour at room temperature prior to mounting onto slides with 4,6-diamidino-2-phenylindole (DAPI)-Prolong Gold (Cat No: P36931; Invitrogen, Carlsbad, CA, USA). Images were collected using the Fluoview FV10-ASW 3.1 Viewer software (Olympus, Tokyo, Japan) with an Olympus FV1000 confocal laser scanning microscope.
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3

Immunofluorescent Profiling of Spinal Cord Microglia

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Rats (n=3 for each group at each time point) were deeply anesthetized and then perfused transcardially with 200 mL cold saline followed by 300 mL 4% cold paraformaldehyde (PFA). Next, lumbar enlargements were collected and fixed in PFA for 8 hours and cryoprotected in 30% sucrose. Samples were further embedded in OCT medium (Torrance, CA, USA) and cut into 8-μm-thick sections. All sections were rinsed in 0.2%Triton X-100 mixed with 5% donkey serum for 40 min at room temperature and then incubated overnight with primary antibodies at 4°C at the following dilutions: mouse anti-CD11b (SM262PS, 1:50, OriGene, MD, US), rabbit anti-CD16 (1:50, Affinity) and rabbit anti-ARG1 (1:100, Affinity). This was followed by incubation with a mixture of Alexa Fluor 594-conjugated AffiniPure Donkey Anti-Rabbit IgG (711-585-152, 1:500, Jackson ImmunoResearch, PA, US) and Alexa Fluor 488-conjugated AffiniPure Donkey Anti-Mouse IgG (715-547-003, 1:500, Jackson ImmunoResearch). After that, sections were washed, stained with DAPI, and sealed. Fluorescence microscopy (Nikon Eclipse E600, Japan was used to capture the images, and Image-Pro Plus 6.0 (MD, US) was used to calculate the intensity).
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4

Identifying rCEnCs via Na+/K+-ATPase and ZO-1

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The expression of Na+/K+-ATPase and ZO-1 were measured to identify rCEnCs on SF and LPA/SF. The histological expression was evaluated after 3 days of culture. The rCEnCs were fixed with 4% formaldehyde (Sigma-Aldrich, USA) at 4 °C overnight and washed with PBS three times. A protein-blocking solution (DAKO, Glostrup, Denmark) was added for 15 min at room temperature to prevent non-specific binding. Fixed samples were incubated with the primary antibodies anti- Na+/K+-ATPase and anti-ZO-1 (1:200, Sata Crux Biotechnology, Dallas, TX, USA) at 4 °C for overnight. Alexa Fluor®594-conjugated AffiniPure Donkey Anti-Rabbit IgG (1:300, Jackson Immuno Research Laboratories, Inc., West Baltimore Pike West Grove, PA, USA) was used as a secondary antibody. The images were taken by confocal laser scanning microscope (LSM 510 META, Zeiss, Oberkochen, Germany) installed in the Center for University-Wide Research Facilities (CURF) at Chonbuk National University.
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5

Establishment of Human Astrocyte and Endothelial Cell Models

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Human astrocytes (HAs) were obtained from Jiamay Biolab (Beijing, China); hCMEC/D3 cell lines were purchased from Jiangyin Yuxi Biotechnology (Jiangsu, China); polyclonal anti-ZO-1 and anti-occludin antibodies were acquired from Biorbyt (Cambridge, UK, cat: orb11587, orb11181); Alexa Fluor 594-conjugated AffiniPure donkey anti-rabbit IgG was obtained from Jackson ImmunoResearch Inc. (West Grove, PA, USA, cat: 711-585-152); human interleukin-1β ELISA kit and human MMP-9 ELISA kit were purchased from Jiamay (Beijing, China, cat: FHK0016, FHK0144); high glucose Dulbecco's Modified Eagle's Medium (DMEM) basal medium was obtained from Hyclone (Logan, UT, United States); and EBM-2 basal medium was purchased from Lonza (Walkersville, MD, USA). Fetal bovine serum (FBS) was obtained from GIBCO (Rockville, MD, USA); phenylmethanesulfonyl fluoride (PMSF) was acquired from Amresco (Solon, OH, USA); the different cocktails were purchased from Yuanye Biotech (Shanghai, China); skimmed milk was obtained from Yili Industrial Group Co. Ltd. (Beijing, China); and anti-β-actin and other materials were purchased from Jiamay Biolab (Beijing, China).
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6

Quantification of NeuN-positive neurons

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The frozen sections obtained above were washed in PBS and rinsed in 0.2% Triton X-100 mixture with 5% donkey serum for 1 h at room temperature. After blocking, each section was incubated with primary antibody rabbit anti-NEUN (1:300; EPR12763, Abcam, Cambridge, UK), followed by Alexa Fluor 594-conjugated AffiniPure Donkey Anti-Rabbit IgG (1:500; 711-585-152, Jackson ImmunoResearch, PA, USA). Next, all sections were washed in PBS and sealed. A fluorescence microscope (Olympus U-HGLGPS, Japan) was used to capture the images, and Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA) was used to calculate the intensity in the selected area of each section.
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