The 4th inguinal mammary glands were harvested from 6-week old and 6-month old virgin female mice. Whole mount preparations were fixed with formalin, stained with carmine alum, and imaged using a Zeiss SteREO Discovery.V12 micoscope. Sections (5 μm) were prepared using tissue fixed in 10% formalin that was dehydrated and embedded in paraffin. A board-certified pathologist examined sections stained with hematoxylin & eosin and imaged using a Zeiss Axiovert 200M. Sections were also stained with antibodies against keratin 5 (BioLegend Cat# 905501 RRID:AB_2565050; 1:50 dilution) and keratin 8 (Developmental Studies Hybridoma Bank Cat# TROMA-I RRID:AB_531826; 1:100 dilution), and immune complexes were detected using AlexaFluor 546 conjugated-goat anti-rabbit IgG (H+L) antibody (Molecular Probes Cat# A11035 RRID:AB_143051) and AlexaFluor 488 conjugated-goat anti-rat IgG (H+L) antibody (Molecular Probes Cat# A11006 RRID:AB_141373) and counterstained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI). Proliferating cells were stained using the endogenous biotin blocking kit (Thermo Fisher Scientific E21390), biotin-conjugated PCNA antibody (Thermo Fisher Scientific Cat# 13-3940 RRID:AB_2533; dilution 1:50), and AlexaFluor 633-conjugated streptavidin (Thermo Fisher Scientific Cat# S-21375 RRID:AB_2313500). Immunofluorescence was examined using a Leica SP2 confocal microscope.
Alexa fluor 633 conjugated streptavidin
Manufactured by Thermo Fisher Scientific
Alexa Fluor 633-conjugated streptavidin is a fluorescent labeling reagent. It consists of the protein streptavidin covalently linked to the Alexa Fluor 633 dye. Streptavidin is a tetrameric protein that binds strongly and specifically to the small molecule biotin.
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4 protocols using alexa fluor 633 conjugated streptavidin
1
Mammary Gland Whole Mount and Histology
2
Immunohistochemical visualization of BDA and GFP
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All flattened tangential sections and series two of the coronal sections were processed to reveal the BDA tracer and to enhance signal from the virus using a 2-day immunohistochemical procedure. On day one, the sections were washed in phosphate buffered saline (PBS; 3 × 5 min), followed by a phosphate buffered saline solution with Triton (PBS 0.1 M, 0.3% Triton, 3% BSA; 2 × 10 min) on a shaker (100 rpm) at room temperature (RT). The sections were incubated with anti-GFP primary antibody (GFP; rabbit anti-GFP, 1:1,000, Thermo Fisher Scientific, A-11122) overnight on a shaker (60 rpm) at 4°C. On day two, the sections were washed in PBS solution (PBS 0.1 M, 0.3% Triton, 3% BSA; 2 × 5 min) and incubated with secondary antibody (Alexa Fluor 488-tagged goat anti-rabbit Ab, 1:1,000, Thermo Fisher Scientific, A-11008) and with Alexa Fluor 633-conjugated Streptavidin (1:400, Thermo Fisher Scientific Cat. No. S-21375, RRID:AB_2313500 ) against BDA on a shaker (60 rpm) at RT (75 min). The sections were rinsed in Tris buffer 0.606% [Tris(hydroxymethyl)aminomethane, pH 7.6; 3 × 10 min], mounted on non-frost microscope slides using a Tris-gelatin solution (0.2% gelatin in Tris-buffer, pH 7.6) and coverslipped with an entellan-xylene solution.
3
Multimodal Fluorescent Labeling and Clearing of Brain Slices
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Brain slices (250 μm thick) containing biocytin-filled neurons and voltron-JF-585 labeling were postfixed in 4% PFA in PB, 0.1 M, pH 7.8) at 4 °C overnight. Slices were repeatedly washed in PB and cleared using CUBIC protocol22 (link). First ‘CUBIC reagent 1’ was used (25 wt% urea, 25 wt% N,N,N′,N′-tetrakis(2-hydroxypropyl) ethylenediamine and 15 wt% polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100) for 1 day at 4 °C. After repeated washes in PB, biocytin was visualized using Alexa Fluor 633-conjugated streptavidin (Thermo Fisher, S21375, 1:1,000) at rom temperature for 3 h. For NeuN staining, primary antibody (Millipore, MAB377, 1:1,000, Mouse, IgG1) was incubated overnight at 4 °C and, after repeated washing with PB, second antibody (Jackson Cy5 AffiniPure Donkey Anti-Mouse IgG (H+L), Code: 715-175-151, 1:500) was incubated for 3 h at room temperature. Slices were then rewashed in PB and submerged in ‘CUBIC reagent 2’ (50 wt% sucrose, 25 wt% urea, 10 wt% 2,20,20′-nitrilotriethanol and 0.1% v/v% Triton X-100) for further clearing. Slices were mounted on Superfrost glass (Thermo Scientific) using CUBIC2 solution and covered with 1.5 mm cover glasses.
4
Visualization of Biotinylated Proteins in HeLa Cells
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HeLa Flp-In T-REx cells were plated on coverslips, and simultaneous induction with tetracycline (1 μg/ml) and treatment with biotin (50 μM) were done 24 h prior to cell fixation with 3.7% formaldehyde in CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM Pipes, pH 6.8). Then, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min before incubation with rabbit polyclonal FLAG antibody (Cell Signaling Technology; catalog no.: 2368) at 1:1000 dilution in the wash buffer (1% bovine serum albumin and 0.1% Triton X-100 in TBS), rinsed five times with wash buffer, and incubated for 30 min with Alexa Fluor 488–conjugated chicken anti-rabbit (Life Technologies; catalog no.: A-21441) at 1:500 dilution together with Hoechst 33342 (Invitrogen; catalog no.: H3570) at 1:10,000 dilution and Alexa Fluor 633-conjugated streptavidin (Thermo Fisher Scientific; catalog no.: S21375) at 1:500 dilution in wash buffer. Cells were washed ten times with the wash buffer and one time with water. Coverslips were mounted with Mowiol. Images were acquired with a Carl Zeiss LSM700 laser scanning confocal microscope (Carl Zeiss MicroImaging) equipped with a plan-apochromat 63×/1.4 numerical aperture objective and operated with ZenBlack 2009.
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