The L4–L5 spinal cord tissues of three rats in each group were selected and fixed in 10% neutral formalin, dehydrated in ethanol, embedded in wax, and then sectioned at a thickness of 4 µm. Spinal cord sections were dewaxed and incubated overnight at 4 °C with CD80 antibody (1:100, bs-2211R, Bioss, Beijing, China) and Iba-1 antibody (1:100, ab283319, Abcam, Cambridge, UK); CD206 antibody (1:100, DF4149, Affinity, Suzhou, China) and Iba-1 antibody (1:100, ab283319, Abcam, Cambridge, UK) were mixed and incubated overnight at 4 °C; C3 antibody (1:100, DF13224, Affinity, Suzhou, China) and GFAP antibody (1:100, ab279290, Abcam, Cambridge, UK) were mixed and incubated overnight at 4 °C; S100A10 antibody (1:100, bs-8503, Bioss, Beijing, China) and GFAP antibody (1:100, ab279290, Abcam, Cambridge, UK) were mixed and incubated overnight at 4 °C. Goat anti-rabbit IgG H&L (Alexa Flour 488,1:200, ab150077, Abcam, Cambridge, UK) and goat anti-mouse IgG H&L (Alexa Flour 647,1:200, ab150115, Abcam, Cambridge, UK) were used as secondary antibodies. Tissue sections were immunofluorescently stained and then observed under a laser confocal microscope (SP8, Leica, Wetzlar, Germany) and photographed. Fluorescence quantitative analysis was performed by Image J software.
Ab279290
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab279290 is a lab equipment product offered by Abcam. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Bs 2211r, by Bioss Antibodies (1 mentions)
Ab283319, by Abcam (1 mentions)
Df4149, by Affinity Biosciences (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab150115, by Abcam (1 mentions)
Bs 0296g fitc, by Bioss Antibodies (1 mentions)
Ab215037, by Abcam (1 mentions)
Hoechst 33342, by Solarbio (1 mentions)
Phospho smad2 smad3, by Cell Signaling Technology (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
Cck 8, by Abcam (1 mentions)
Rpe 65, by Thermo Fisher Scientific (1 mentions)
β actin rabbit, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab8878, by Abcam (1 mentions)
Ab228551, by Abcam (1 mentions)
Alexa fluor 555 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Ab8227, by Abcam (1 mentions)
7 protocols using ab279290
1
Immunofluorescent Staining of Rat Spinal Cord
2
Immunofluorescent Analysis of NSC Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 4% paraformaldehyde for 15-30 min at room temperature. After washing with PBS for three times, the cells were covered with 4% BSA solution containing 0.1% Triton X-100 at room temperature for 1 h and then incubated overnight with 4% BSA-diluted primary antibodies β-tubulin III (1:500, ab215037; Abcam) and GFAP (ab279290, 1:50; Abcam). The samples were removed from the 4˚C condition on the second day, reheated at room temperature for 30 min and then incubated with goat anti-rabbit IgG Cy5 (1:500, bs-0295G-Cy5; Bioss) or goat anti-mouse IgG FITC (1:500, bs-0296G-FITC; Bioss) at room temperature for 1 h. The solution was then discarded, and the samples were washed with PBS for three times. Nuclei were stained at room temperature with Hoechst 33342 (C0030; Beijing Solarbio Science & Technology Co., Ltd.). The NSCs differentiation were identified by detecting the expression of β-tubulin III and GFAP under a fluorescence microscope (scale bar, 20 µm; Zeiss AG).
3
Immunostaining of Cellular Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were applied: Tetraspanin-4 (NBP1-59438, Novus); RPE 65 (MA1-16578; Thermo Fisher Scientific); GFAP (ab279290, Abcam); CD11b (ab8878, Abcam); integrin α5 (ab288767, Abcam); EOGT (ab190693, Abcam); NDST1 (26203-1-AP, Proteintech); Alix (2171, Cell Signaling); Alix (92,880, Cell Signaling); Smad2/3 (3102, Cell Signaling); Phospho-Smad2/Smad3 (8828, Cell Signaling); Alpha-Smooth Muscle Actin (MA5-11547, Thermo Fisher Scientific); GAPDH (5174, Cell Signaling) and β-actin Rabbit antibodies (ab8227, Abcam). Secondary antibodies used included the following: Alexa Fluor 488 Anti-Mouse; Alexa Fluor 555 Anti-Rabbit; Alexa Fluor 555 Anti-Mouse; and Alexa Fluor 555 Anti-Rabbit (Thermo Fisher Scientific). Additionally, CCK8 (ab228551, Abcam), Dulbecco’s modified Eagle’s medium (DMEM)/F12 culture media, and fetal bovine serum were obtained from Thermo Fisher Scientific.
4
Immunohistochemical Analysis of Sertad1 Expression in Murine Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were anesthetized 48 h after reperfusion, transcardially perfused with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde. Their brains were rapidly removed and postfixed with 4% paraformaldehyde. Brains were then immersed in 20% and 30% sucrose overnight at 4°C, embedded in Tissue-Tek OCT compound (Sakura, Japan), and subjected to section on a freezing microtome. The sections (10 μm) were washed with PBS and blocked in QuickBlock™ immunostaining blocking solution (Beyotime, China) for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: rabbit anti-Sertad1 (1:200, ARP34309_T100; AVIVA, USA), mouse anti-NeuN (1:100, 66836-1-Ig; Proteintech, China), mouse anti-GFAP (1:200, ab279290; Abcam, USA), mouse anti-Iba1 (1:500, GT10312; GeneTex, USA). After washing, the sections were incubated with Alexa Fluor Plus 488 goat anti-rabbit secondary antibody (1:200, A32731; Thermo Fisher Scientific, USA) or Alexa Fluor Plus 555 goat anti-mouse secondary antibody (1:200, A32727; Thermo Fisher Scientific) at 37°C for 1 h. DAPI (Beyotime) was used to stain cellular nuclei at 37°C for 10 min. Finally, the sections were photographed using a microscope with a digital camera (Olympus, Japan).
5
Immunofluorescence Staining of Neural Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The procedure for immunofluorescence staining is described in previous studies.19 The following primary antibodies were used: GFAP (1:500, rabbit, cat # ab279290, Abcam); PSD‐95 (1:500; mouse, MA1‐046, Thermo); Megf10 (1:200; rabbit, 32160702, Sigma); LAMP2 (1:200; rabbit, YT5711, Imunoway). Tissues were incubated at 4°C for 2 days, followed by incubation with the corresponding secondary antibodies of Alexafluor 488 or 594 (1:500, Abcam) for 1 hour at room temperature. Samples were mounted using Vectashield containing DAPI for cell nuclei staining (ab104139, Abcam).
6
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell suspension was centrifuged at 300 x g for 5 min at room temperature, and the supernatant was discarded, total protein was extracted from cells using lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology) with protease inhibitors PMSF(ST506; Beyotime) and kept on ice for 30 min. Protein concentrations were determined by Bradford assay and then 20 µg each protein sample were boiled for 5 min, separated by 10% SDS-PAGE, transferred onto a PVDF membrane, blocked with 5% skim milk in TBST buffer (20% Tween) for 3 h at room temperature and then incubated with primary antibodies at 4˚C overnight. Primary antibodies were Nestin (1:1,000, ab6320; Abcam), Sox2 (1:1,000, ab171380; Abcam), GFAP (1:1,000, ab279290; Abcam), Map2 (1:1,000, ab281588; Abcam), ERRα (1:1,000, bs-6998R; Bioss), TGF-β1 (1:1,000, ab215715; Abcam), α-tubulin (1:10,000, ab7291; Abcam), AURKB (1:10,000, ab45145; Abcam) and Id2 (1:500, ab90055; Abcam). The membranes were then washed three times with TBST and incubated with a secondary antibody (1:30,000, Goat Anti-Mouse IgG H&L/HRP, bs-40296G-HRP; 1:30,000, Goat Anti-Rabbit IgG H&L/HRP antibody, bs-40295G-HRP; Bioss) at room temperature for 1 h. Protein bands were visualized using ECL (MilliporeSigma).
7
Immunofluorescent Staining of Brain Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were anesthetized at 72 h after I/R and perfused with PBS and 4% paraformaldehyde. The brain was removed from each animal and fixed with 4% paraformaldehyde overnight, transferred to 20 and 30% sucrose, and cut into 5 μm-thick sections on a freezing microtome. After washing and blocking, the sections were incubated overnight at 4°C with the following primary antibodies: anti-Rab20 (1:200; 11616-1-AP, Proteintech Group, Inc., Wuhan, China), anti-NeuN (1:100; 66836-1-Ig, Proteintech Group, Inc.), anti-IBA1 (1:500; 011-27991, Fujifilm Wako, Japan), and anti-GFAP (1:200; ab279290, Abcam, Cambridge, MA, USA). After washing, the sections were incubated with donkey anti-rabbit Alexa fluor 488 secondary antibody (1:500; ab150073, Abcam), donkey anti-rabbit Alexa fluor 555 secondary antibody (1:500; A31572, Thermo Fisher Scientific, MA, USA), donkey anti-mouse Alexa fluor plus 555 secondary antibody (1:500; A32773, Thermo Fisher Scientific, MA, USA), and donkey anti-goat Alexa fluor plus 555 secondary antibody (1:500; A32816, Thermo Fisher Scientific) for 2 h at room temperature in the dark. Finally, the nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI) for 15 min.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!