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).
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.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (2 mentions)
Dfc7000 t camera, by Leica (1 mentions)
Application suite x software, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluorsave, by Merck Group (1 mentions)
Prolong gold dapi, by Thermo Fisher Scientific (1 mentions)
Fluoview fv10 asw 3.1 viewer software, by Olympus (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Alexa fluor 488 conjugated affinipure donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Image pro plus 6, by Nikon (1 mentions)
Protein blocking solution, by Agilent Technologies (1 mentions)
Ebm 2 basal medium, by Lonza (1 mentions)
Phenylmethanesulfonylfluoride pmsf, by Avantor (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Epr12763, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
U hglgps, by Olympus (1 mentions)
6 protocols using alexa fluor 594 conjugated affinipure donkey anti rabbit igg
1
Immunofluorescence Analysis of Lung Histology
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.
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).
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.
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).
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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