Embryos were collected, fixed with 4% PFA, washed in PBST (1% BSA, 1% DMSO, 0.1% Triton X-100 in PBS, pH 7.4), blocked in 10% normal donkey or goat serum/PBDT, and incubated overnight at 4°C with a primary antibody targeting acetylated α-Tubulin (acTub; 1:1000; Sigma-Aldrich, T7451). Further PBST washes and blocking were followed by secondary antibodies (Invitrogen) overnight at 4°C. DAPI was added to stain nuclei. After further PBDT and PBS washes, embryos/larvae were mounted to image the left olfactory epithelium in each sample. Confocal microscopy was performed using a Zeiss LSM 800 microscope with a 40x/1.1 W objective.
Actub
Manufactured by Merck Group
AcTub is a laboratory equipment product from the Merck Group. It is designed for the centrifugation of samples in a controlled environment. The core function of AcTub is to separate and isolate components within a liquid sample through the application of centrifugal force.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 800 microscope, by Zeiss (1 mentions)
Observer z1 microscope, by Zeiss (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Bz 9000 fluorescence microscope, by Keyence (1 mentions)
Alexa fluor 546 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
4 protocols using actub
1
Immunohistochemistry of Olfactory Epithelium
2
Microtubule Integrity Assessment in Axons
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The integrity of microtubules was assessed by immunostaining with antibodies against acetylated tubulin (AcTub; Sigma-Aldrich) and tyrosine hydroxylase (TH) (Pel-Freeze Biological, Rogers, AR) after treatment with 6-OHDA in the axonal compartment. Axons with three AcTub breaks or more were considered damaged and the number as a percentage of total axons in TH-positive and negative axons was determined.
3
Immunocytochemical Detection of Cilia
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Cells were fixed with 4% PFA in 5% sucrose. Immunocytochemistry was performed with a monoclonal primary antibody against acetylated tubulin (AcTub, Sigma-Aldrich, Cat# T6793 or Cat# T7451). A DyLight 405-labelled goat anti-mouse IgG (ThermoFisher, Cat# 35501BID) or an Alexa Fluor 647-labelled donkey anti-mouse IgG (ThermoFisher, Cat#A31571) were used as secondary antibodies. Images were acquired and analysed as described above for living cells or with a Z.1 Observer microscope (Zeiss, Oberkochen, Germany). Perinuclear structures positive for acetylated tubulin were considered cilia when >0.5 µm in length.
4
Immunofluorescence Assay for Primary Cilia
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The cells grown on Lab‐Tek plates or coverslips were fixed with 3.7% formaldehyde in PBS for 15 min. After two washes with PBS, the cells were permeabilized with 0.1% Triton X‐100, transferred into a blocking solution (20% goat serum in PBS) for 30 min, and incubated with mouse anti‐acetylated tubulin (Ac‐tub) (T7451; Sigma‐Aldrich; 1:2000) and rabbit anti‐GFP (598; MBL; 1:2000) primary antibodies for 16–24 h at 4°C. The bound antibodies were detected using appropriate secondary antibodies (Alexa Fluor 546‐conjugated goat anti‐mouse IgG or Alexa Fluor 488‐conjugated goat anti‐rabbit IgG; Life Technologies Co.).45 (link) Because primary cilium in vitro cell culture is usually lying flat along the coverslip, the length from a two‐dimensional image was measured using a line measurement tool PhotoRuler Ver. 1.1 software (The Genus Inocybe, Hyogo, Japan) under the BZ‐9000 fluorescence microscope (Keyence). Data for at least 100 cilia per treatment were obtained from at least three independent experiments, and the values are presented as means ± SEM.
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