Q&A Apr 15, 2026

Detailed protocol for Johnsen scoring in testicular histopathology used in our study  BackgroundThe Johnsen scoring system is a semiquantitative histopathological method used to evaluate spermatogenesis in seminiferous tubules. It assigns a score from 1 to 10 according to the most advanced germ cell type present and the degree of spermatogenic maturation. Higher scores indicate more advanced spermatogenesis, and a score of 8 or higher generally indicates the presence of spermatozoa.SpecimensTest...

Q&A Apr 14, 2026

AbstractVoltage-gated ion channels such as hERG (KV11.1) adopt multiple conformational states that are critical for ion conduction and drug binding, yet experimentally resolved structures often capture only a limited subset of these states. Here, we describe a protocol for generating hERG channel models in closed, open, and inactivated conformations using ColabFold AlphaFold2 notebook with template-guided sampling (1). The approach uses carefully constructed structural templates derived from hom...

Q&A Apr 13, 2026

Human hPMBC isolation1. For each tube of blood, prepare equal volume as there is blood of Ficoll into a 50mL Falcon tube. Note, try to process the blood onto Ficoll as soon as possible after the collection.2. Dilute blood in equal volume of HBSS (i.e. 1:2 dilution).3. Carefully overlay the diluted blood using a 10mL serological pipette by holding the 50mL Falcon tube containing the Ficoll at a 45º angle slowly pipette out so that there is minimal disturbance to the interface.4. Centrifuge at 500...

Q&A Apr 10, 2026

This protocol was used in the work “Investigating the composition and recruitment of the mycobacterial ImuA′–ImuB–DnaE2 mutasome” (DOI: 10.7554/eLife.75628) to purify the Mycobacterium smegmatis β clamp (DnaN) for biochemical experiments.1. ExpressionExpress the dnaN gene from Mycobacterium smegmatis (DNA polymerase III β subunit) in E. coli BL21(DE3) cells for 16 hours at 16 °C. In this paper, the expression vector pETNKI-his-3C-LIC-kan from the NKI-LIC vector suite (Luna-Vargas et al. 2011, DO...

Q&A Apr 8, 2026

Protocol of yeast one hybrid assay using pLacZi2u/pJG4-5 systemVectorspLacZi2μ (AmpR in E.coli; Ura- in yeast) and pJG4-5 (or named pB42AD; AmpR in E.coli; Trp- in yeast) plasmids. 2μ origin of replication was PCR amplified from pEG202 and inserted into the HindIII and EcoRI sites of the reporter plasmid pLacZi (Clontech), resulting in pLacZi2μ vector.The coding sequence of DNA binding protein should be inserted into the MCS of pJG4-5 to generate the AD-fusion construct and the cis-element to be...

Q&A Apr 6, 2026

Cauda sperm collection protocolDissect out cauda epididymis from an adult mouse and transfer to a 35mm dish containing 1ml of pre-warmed Whitten’s Media (100 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM Glucose, 1 mM Pyruvic acid, 4.8 mM Lactic acid (hemicalcium), and HEPES 20 mM). Gently squeeze the epididymis to allow the caudal fluid to ooze out in the dish.  Incubate at 37°C for 15 minutes After incubation transfer the sperm containing media to a fresh tube and incubate for anoth...

Q&A Mar 31, 2026

Surgical Denervation of Interscapular Brown Adipose Tissue (iBAT) MaterialsAnesthetic agentSyringeInjection needleSterilized surgical instrument setsStraight forcepsCurved forcepsScissorsNote: To prevent contamination, it is recommended to use separate instrument sets for skin incision and for deeper tissue dissection. Instruments should be sterilized before use in different animals.Sutures: Pronova (blue, 4-0, 90 cm, taper-cut needle, 26 mm, strong curvature; 3975H)5% Hibitane (chlorhexidine; d...

Q&A Mar 30, 2026

Rat Lung scRNAseq DissociationLast updated 2026-03-30 by MSBRRaredon Lab Animals: SD Female and Male ~220/250grams Anesthetic: KX 75mg/5mg per kg body weight Pre-Op: Intraperitoneal heparin 0.15ml at 1000U/ml (critical to allow blood clearance from lung tissue) Setting: Biosafety hood for sterile cell isolation. Benchtop OK for non-sterile. Surgery:Abdominal incisionDiaphragm incision and tensionChest clamshellThymic dissection to expose great vesselsNeck dissection to expose tracheaCannulate tr...

Q&A Mar 23, 2026

Protocol: Investigating the Mechanism of MFN2 in Regulating Mitochondria-Associated Membranes (MAMs) and Cardiolipin in Vascular Dementia AbstractVascular dementia (VaD) is a progressive cognitive impairment caused by cerebrovascular disease, with mitochondrial dysfunction playing a pivotal role in its pathogenesis. Mitofusin 2 (MFN2) is a key protein mediating the tethering between the endoplasmic reticulum (ER) and mitochondria at specific contact sites known as mitochondria-associated membran...

Q&A Mar 18, 2026

Soil Extraction Plating for the Detection of Aspergillus terreus from Environmental SamplesAuthors and AffiliationsJan Schobert¹, Paul Illmer², Roya Vahedi-Shahandashti¹*, Cornelia Lass-Flörl¹*¹ ISHAM Working Group of Aspergillus terreus, ECMM Excellence Center of Mycology, Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Austria² Institute of Microbiology, University of Innsbruck, AustriaAbstractThis protocol describes a selective soil extraction plating method fo...