Journal: STAR protocols
Article Title: Protocol for identifying protein synthesis activity in specific cell types of the testicular lumen.
doi: 10.1016/j.xpro.2025.103661
Figure Lengend Snippet: Figure 4. Expected outcomes of detecting new synthetic proteins in the testicular lumen (A) Whole-mount immunofluorescence analyses of 4-day-old control mice testes treated with or without 100 mM cycloheximide (CHX). Nascent protein synthesis was detected by staining with OPP-594 (red); spermatogonia was detected by staining with the germ cell marker DDX4 (green). Scale bars, 20 mm n = 3. (B) Quantitative analyses of protein synthesis efficiency in spermatogonia treated with or without 100 mM cycloheximide (CHX). Student’s t test; n = 3.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-DDX4 antibody (1:200) Abcam Cat# ab27591; RRID: AB_11139638 Goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 (1:500) Thermo Fisher Scientific Cat# A-11001; RRID: AB_2534069 (Continued on next page) 2 STAR Protocols 6, 103661, March 21, 2025 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Cycloheximide (CHX) Cell Signaling Technology Cat# 2112 BlockAid blocking solution Thermo Fisher Scientific Cat# B10710 Image-iT fixative solution Thermo Fisher Scientific Cat# R37814 Triton X-100 Sigma Cat# 93443 Phosphate-buffered saline (PBS, pH 7.4) Gibco Cat# 10010023 Minimum essential medium a (MEMa) Gibco Cat# 12561056 Glial cell line-derived neurotrophic factor (GDNF) PeproTech Cat# 450-10 Basic fibroblast growth factor (bFGF) PeproTech Cat# 100-18B Insulin Sigma-Aldrich Cat# I 2643 Bovine serum albumin(BSA) MP Biomedicals Cat# 810661 Fetal bovine serum (FBS) HyClone Cat# SH30396.03 Lipoprotein Sigma-Aldrich Cat# L4646 GlutaMAX Gibco Cat# 35050061 N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) Gibco Cat# 15630-080 Non-essential amino acids (NEAA) Gibco Cat# 11140-050 Sodium selenite Sigma Cat# S5261 Putrescine Sigma Cat# P5780 2-methoxyethanol (2-ME) Sigma Cat# M3145 Transferrin Sigma Cat# T0665 Ascorbic acid Sigma Cat# M4544 Critical commercial assays Click-iT Plus OPP Alexa Fluor 594 protein synthesis assay kit Thermo Fisher Scientific Cat# C10457 Experimental models: Organisms/strains Mouse; C57BL/6J, 4-days-old, male mice Speford Biotechnology Company (Beijing, China) N/A Software and algorithms ZEN lite version 2 Zeiss https://www.zeiss.com/microscopy/zh/ products/software.html ImageJ v.1.8.0 ImageJ software https://imagej.net/ij/ GraphPad Prism v.8 GraphPad Software https://www.graphpad.com/ Other 96-well culture plates Costar 3599 Confocal microscope Zeiss LSM 780 Rocking lab shaker Bolaiyan BLY-100E Adhesion microscope slides Citotest 188105 Microscope cover glasses BRAND GMBH + CO KG 470820 ll OPEN ACCESSProtocol
Techniques: Control, Staining, Marker
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