T cells migrating within the collagen gel matrix were fixed with prewarmed 4% paraformaldehyde (Electron Microscopy Sciences) in PBS. The collagen gel containing the cells was washed in PBS, permeabilized with 0.5% Triton X-100, blocked with 2% bovine serum albumin (Sigma-Aldrich) in 0.1% Triton X-100, and incubated with primary antibodies for 1 h at 4°C. Anti–α-Tubulin–Alexa Fluor 488 (Life Technologies) was used with anti-pericentrin antibody (Abcam), followed by Alexa Fluor 647–conjugated anti–rabbit secondary antibody (Life Technologies). Hoechst dye 33342 at 1 µg/ml (Life Technologies) was also used. Stained gels were kept in PBS at 4°C for no longer than 24 h before image capture. Imaging was performed on a Leica SP8 or SP5 white light laser confocal microscope with a 63× glycerol immersion objective lens, using an image stack vertical step setting of 0.15–0.2 µm. Where indicated, after staining gels were physically compressed to facilitate simultaneous visualization of all vertical stacks.
Anti pericentrin antibody
Manufactured by Abcam
Anti-pericentrin antibody is a laboratory reagent used to detect and study the pericentrin protein in biological samples. Pericentrin is a structural component of the centrosome, which is an important organelle involved in cell division and microtubule organization. This antibody can be used in various experimental techniques, such as immunoblotting, immunohistochemistry, and immunocytochemistry, to investigate the localization and expression of pericentrin in cells and tissues.
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Lab products found in correlation
Mouse igg blocking reagent, by Vector Laboratories (2 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
Tcs sp2 spectral confocal microscope, by Leica (2 mentions)
Anti rabbit cy3, by Jackson ImmunoResearch (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Recombinant human il 6, by R&D Systems (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti p stat3, by Cell Signaling Technology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti s6, by Cell Signaling Technology (1 mentions)
Anti ps6, by Cell Signaling Technology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti arl13b, by Proteintech (1 mentions)
Zenon alexa fluor 647 rabbit igg labelling kit, by Thermo Fisher Scientific (1 mentions)
Mouse anti acetylated α tubulin, by Merck Group (1 mentions)
5 protocols using anti pericentrin antibody
1
Visualizing T Cell Migration in 3D
2
Quantifying Centrosome Dynamics in Hepatocytes
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Liver cryosections (5μm) were fixed with 4% paraformaldehyde and blocked for 1 hour with mouse IgG blocking reagent (vector labs). After blocking with Donkey serum (5% in PBS-0.3%Tween), sections were incubated with 1/500 anti-pericentrin antibody (Abcam) overnight. Secondary 1/500 Anti-rabbit Cy3 (Jackson immunoresearch, stratech) and 1/500 Alexafluor 488 –phalloidin (invitrogen) was then applied on the sections for 1-2hours. Sections were mounted with vectashield plus DAPI medium. Sections were viewed and analysed using a Leica TCS SP2 spectral confocal microscope and Leica confocal software (Leica, Heidelberg, Germany). Centrosomes numbers were counted in at least 150 nuclei of hepatocytes from each mouse for each genotype (N=3-5) at D4 and D6.
3
Quantifying Centrosome Dynamics in Hepatocytes
4
Antibody Reagents and Inhibitors for Signaling Studies
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Anti-pericentrin antibody (cat# ab4448) was purchased from Abcam, anti-myc (clone 4A6, cat# 05–724) from Millipore, anti-GFP (clone JL-8, cat# 8371–1) from Clontech, and anti-actin (cat# A 5441) and anti-FLAG (cat# F4042) were purchased from Sigma. Anti-Erk (cat# 4696), anti-pErk (cat# 4370), anti-STAT3 (cat# 9139), anti-pSTAT3 (cat# 9145), anti-S6 (cat# 2317), and anti-pS6 (cat# 4858) were purchased from Cell Signaling. The anti-PC1 C-terminal antibody has been previously described [12 (link)]. Doxycycline was purchased from Sigma Aldrich. CaM agarose (Sepharose 4B) was purchased from GE Healthcare. Recombinant human IL6 was purchased from R&D Systems. W12-HCl was from Sigma and W13-HCl was from Tocris.
5
Immunofluorescence Labeling of Cilia Components
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URECs were xed in ice-cold methanol for 10 min. After 30 min saturation with 5% BSA in PBS, cells were incubated for 1 h at room temperature with the following primary antibodies in blocking solution: rabbit anti-ARL13B (Proteintech, 17711-1-AP); mouse anti-acetylated α-tubulin (Sigma T6793). Following washes in PBS, cells were incubated at room temperature for 1 h with the following secondary antibodies: donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher); donkey anti-mouse Alexa Fluor 594 (Thermo Fisher). Following further washes in PBS, cells were incubated overnight at 4 °C with primary rabbit anti-Pericentrin antibody (Abcam ab 4448) directly labelled using Zenon Alexa Fluor 647 rabbit IgG labelling kit (Thermo Fisher), washed with 0.1% PBS Tween and post-xed with 4% PFA for 15 min. Following nal washes in PBS, cells were mounted in Vectashield (Vector Laboratories Ltd, H-1200). Images and z-stacks were captured in a blinded fashion, using a Nikon (A1) confocal inverted microscope.
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