Mice were anesthetized and transcardially perfused with 0.9% saline and 4% formaldehyde, respectively. The brains were carefully removed, immediately dehydrated with 15% sucrose, and immersed in 30% sucrose the next day. Coronal brain sections of 10 μm thickness were incubated with 1% Triton X-100 for 30 min at room temperature. After blocking with 5% bovine serum albumin for 1 h at 37°C, sections were incubated overnight at 4°C with anti-GFAP mouse antibody (A00213, BOSTER, Inc.1:300), anti-NeuN mouse antibody (MAB377, Millipore, USA, 1:50) and anti-Iba-1 Chicken antibody (Cat234006, SYSY, Inc., Germany, 1:400). Sections were then incubated with Alexa Fluor@647-conjugated goat anti-chicken IgY (ab150171, Abcam, 1:400), Alexa Fluor 594-conjugated goat anti-mouse IgG (bs-0296G-AF594, Bioss, 1:300), or Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L; SA00006-1, Proteintech, 1:300) and incubated with DAPI (Sigma, USA, 1:200) to stain nuclei. All images were taken with an A1 + R laser confocal microscope (Nikon, Tokyo, Japan).
Sa00006 1
Manufactured by Proteintech
Sourced in United States
SA00006-1 is a laboratory equipment product. It serves a core function within the research process, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
A1r laser confocal microscope, by Nikon (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Sa00003 3, by Proteintech (2 mentions)
Ab150171, by Abcam (1 mentions)
Bs 0296g af594, by Bioss Antibodies (1 mentions)
Ab6142, by Abcam (1 mentions)
Sa00013 4, by Proteintech (1 mentions)
P0096, by Beyotime (1 mentions)
Sa00013 3, by Proteintech (1 mentions)
Sa00006 2, by Proteintech (1 mentions)
Ab184699, by Abcam (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by Proteintech (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Sa00006 4, by Proteintech (1 mentions)
Sa00006 8, by Proteintech (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Proteintech (1 mentions)
5 protocols using sa00006 1
1
Multimodal Immunofluorescence Imaging of Mouse Brain
2
Fluorescence Immunocytochemistry of Nestin in NSCs
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Cell cultures in 96-well plates were examined by fluorescence immunocytochemistry. The cells were washed twice in PBS and then fixed in 4% paraformaldehyde for 30 min at room temperature. The cells were then washed twice with PBS and permeabilized with 0.3% Triton X-100 for 20 min. Non-specific binding sites were blocked via incubation with 3% goat serum for 40 min. The cells were then incubated with a mouse monoclonal primary antibody against nestin (a marker of NSCs) (ab6142; Abcam) at a dilution of 1:200 in 1% BSA overnight at 4°C. On the following day, the cells were washed 3 times for 5 min each time and incubated with rhodamine-conjugated goat anti-mouse secondary antibody (SA00006-1; Proteintech, Chicago, IL, USA) at room temperature for 1.5 h in the dark; this was followed by 2 washes in PBS. Subsequently, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and the stained cells were observed under a fluorescence microscope (Olympus AX80; Olympus).
3
Visualizing Glial Cells and Microglia in Rat Brain
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Immunofluorescence was performed by a method described previously [29 (link)]. After anesthesia, 0.9% saline and 4% formaldehyde were transcardially infused into rats. The brains were carefully removed and dehydrated by 15% and 30% sucrose. Rats were anaesthetized and transcardially perfused with 0.9% saline and 4% formaldehyde. The brains were removed carefully and dehydrated with 15% sucrose and 30% sucrose. Ten-micrometer-thick coronal brain sections were harvested and incubated with 1% Triton X-100 for 30 min at room temperature. Subsequently, the sections were blocked for 1 h at 37°C with 5% bovine serum albumin and then incubated with anti-GFAP mouse antibody (A00213, BOSTER Co.) and anti-Iba-1 goat antibody (NB100-1028, Novus Co., USA, 1 : 200) at 4°C overnight. The next day, the sections were incubated with FITC-conjugated AffiniPure donkey anti-goat IgG (H+L; SA00003-3, Proteintech, 1 : 200) or Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L; SA00006-1, Proteintech, 1 : 200). The DAPI was used to stain nuclei (Sigma, USA, 1 : 200). The images were taken using an A1+R laser confocal microscope (Nikon, Tokyo, Japan).
4
Immunofluorescence Analysis of Autophagy Markers
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OT‐I CTLs were treated with DMSO or UA for 16 h followed by fixation for 15 min with 4% paraformaldehyde (PFA) (15 710, Electron Microscopy Science) at room temperature. Cells were permeabilized with permeabilization buffer (Triton X‐100, P0096, Beyotime) for 5 min at room temperature and then blocked with 30% Normal Goat Serum (SL038, Solarbio) for 30 min at room temperature. Cells were stained at 4 °C overnight with the following primary antibodies: anti‐LC3I/II (1:200, PM036, MBL), anti‐ERK1/2 (1:200, ab184699, Abcam), anti‐p‐ERK (1:200, 4377s, Cell Signaling Technology), anti‐ULK1 (1:100, sc‐390904, Santa), anti‐Atg16 (1:200, PM040, MBL). The samples were washed twice with TBST and incubated for 1 h at room temperature with the following secondary antibodies: anti‐rabbit Alexa Fluor plus 488 (1:2,00, SA00006‐2, Proteintech), anti‐rabbit Alexa Fluor plus 594 (1:200, SA00013‐4, Proteintech), anti‐mouse Alexa Fluor plus 488 (1:200, SA00006‐1, Proteintech), anti‐mouse Alexa Flour 594 (1:200, SA00013‐3, Proteintech), and DAPI (C1002, Beyotime). Images were acquired with Fast Airyscan LSM900 Confocal microscope (Zeiss) with 60× oil objective and analyzed using Zen2010 software.
5
Immunofluorescence Staining of Brain Sections
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The slides of brain sections were fixed with 4% formaldehyde solution for 30 min at room temperature, incubated with 1% Triton X-100 for 30 min, and blocked with 5% goat or donkey serum for 1 h at 37 °C. Subsequently, sections were incubated overnight at 4 °C with the following primary antibodies: OTULIN (bs-14689R, Bioss Co., Beijing, China, 1:50), NeuN (MAB377, Millipore Co., Germany, 1:200), MAP2 (4542, Cell Signaling Technology, USA, 1:100) or Iba-1 (NB100-1028, Novus Co., USA, 1:200). After washing three times with PBS, sections were reacted with the following fluorescent secondary antibodies at 37 °C for 1 h: Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L; SA00006-4, Proteintech, 1:200), Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L; SA00006-1, Proteintech, 1:200), FITC-conjugated AffiniPure donkey anti-goat IgG (H+L; SA00003-3, Proteintech, 1:200), and 594-conjugated AffiniPure donkey anti-rabbit IgG (H+L; SA00006-8, Proteintech, 1:200). DAPI (Sigma, USA, 1:200) was used to stain cellular nuclei at 37 °C for 10 min. All images were observed and acquired using an A1+R laser confocal microscope (Nikon, Tokyo, Japan).
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