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A preprint repository for life science protocols
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Animals
Q&A Dec 24, 2025Biological Electron Microscopy Sample Preparation Protocol for Rabbit Corneal Tissue1. Animal Tissue Sample Collection:New Zealand rabbits were euthanized, and corneal tissues from different groups were collected. Sampling was performed within 1-3 minutes post-euthanasia. Corneal tissue samples were trimmed to approximately 2mm × 2mm in size and made as thin as possible. If immediate trimming was not feasible, samples were first placed in electron microscopy fixative for approximately half an ho...
Production of Nanobody 35
Q&A Dec 21, 2025AbstractNanobody 35 (Nb35) binds the heterotrimeric G protein complex and stabilizes it against GTPγS-induced dissociation. This nanobody is widely employed in GPCR structural studies. Here, we give details of its recombinant production. Materials and reagents1. E. coli BL21 (DE3) cells (TIANGEN, China Catalog number: CB105-02).2. Plasmids encoding Nb35 protein.3. TB culture medium with 100 mg/mL ampicillin (Sangon Biotech, China, catalog number: A100339-0025), 2 mM MgCl2 (Sinopharm Chemical Rea...
Co-immunoprecipitation and western blotting
Q&A Dec 18, 2025Crude cell extraction from Drosophila Upd-tumor testes Materials:1M Tris-HCl (pH8.0)5M NaCl0.5M EDTA (pH8.0)50% Glycerole10% NP-40PhoSTOP (Roche, cat No: xxx; store at -20˚C)EDTA-free Protease Inhibitor cocktail (Roche, cat No: xxx; store at -20˚C)300 mM PMSF stock in 100% isopropanol (store at RT)Lysis buffer [20 mM Tris-HCl (pH8.0), 100 mM NaCl, 2 mM EDTA, 10% Glycerole, 0.2% NP-40,1x solution of PhoSTOP cocktail (Roche), 1x solution of cOmplete EDTA- free protease inhibitor cocktail (Roche)...
Direct co-culture of parasites
Q&A Dec 11, 2025Staining with CellTracker Red CMTPX Dye-Prepare the working solution starting from the 1 mM stock. Dilute 1 µL of the stock solution in 1 mL of serum-free culture medium to obtain a final concentration of 1 µM (adjust volumes according to the number of cells to be stained).-Resuspend the cell pellet in the required volume of the 1 µM dye-containing medium. Incubate the cells for 20 minutes at 37 °C, protected from light.-Wash the cells three times with PBS, centrifuging between each wash. For Tr...
Protocol for the Relative Water Content Determination, Proline Content Analysis and Field Capacity Measurement to Observe Drought Stress in Capsicum chinense Jacq (Umorok) Treated with Three Levels of Biochar
Q&A Dec 6, 2025Abstract: This study investigates the effects of three biochar levels (120 g, 300 g, and 420 g per plant) on proline content and relative water content (RWC) in Capsicum chinense Jacq. under field capacity of 45%. Leaf discs (2 × 2 cm; 0.50 g fresh weight) from mature, fully expanded leaves were collected in zipper-locked bags, transported on ice, and weighed to obtain fresh weight. Samples were then hydrated in distilled water for 4 h to determine turgid weight, followed by drying in a hot air ...
Nanobody screening by phage display
Q&A Dec 3, 2025Protocol for Amplification of Phage-Displayed Nanobody Library The stock vial we distributed contains 1.6 mL of phage glycerol stock of the nanobody library, with a titer of 2.2 × 1011 cfu/mL. The stock should be stored at -80°C. The diversity of the nanobody library is 2.4 × 1010. To maintain the integrity of the library during amplification, and considering that E. coli reaches a density of approximately 5 × 108 cells/mL at OD600 ~0.6, we recommend using at least 200 mL of bacterial culture fo...
Synthesis of thanatin and derivatives 1 to 7
Q&A Dec 2, 2025Synthesis of thanatin and derivatives 1-7Peptides were synthesized by solid phase peptide synthesis (SPPS) based on a mixed solid/solution phase procedure using the Fmoc/tBu strategy.Fmoc-Tyr(tBu)-OH or Fmoc-Nle-OH or Fmoc-D-Tyr(tBu)-OH was loaded onto 2-chlorotritylchloride resin in dichloromethane in presence of N,N-diisopropylethylamine (DIEA).After capping (MeOH), a typical loading of 0.4–0.8 mmol/g was achieved. The peptide was assembled by standard automated Fmoc SPPS on 0.025 mmol scale (...
Maintenance of Drosophila S2 cells and CO2 exposure
Q&A Nov 24, 2025Maintenance of Drosophila S2 Cells and CO2 ExposureCell culture conditionsDrosophila S2 cells were maintained at room temperature and protected from light in Schneider’s Insect Medium supplemented with 10% fetal bovine serum (FBS; Valley Biomedical) and 0.2% Penicillin-Streptomycin (Gibco). Cells were cultured under standard non-CO2 incubator conditions. Plate preparation for cell attachmentSix-well plates were pre-treated to promote S2 cell attachment. Wells were incubated with 1 N HCl for 1 h ...
In vitro culture of adult mouse DRG neurons
Q&A Nov 24, 2025This protocol is a detailed description of the method used in the research article Multimodal control of dendritic cell functions by nociceptors, published in Science, 2023. 379, eabm5658 Materials and reagentsPoly-D-LysineLamininPBSLeibowitz 15 mediumPercoll Digestion solution 1 (filter-sterilized): 60U papain 0.5 mM EDTA 1.5 mM CaCl2 in HEPES-buffered HBSS Digestion solution 2 (filter-sterilized): 4mg/ml Collagenase type-2 5mg/...
Cryopreservation
Q&A Nov 13, 2025Cryopreservation and sample preparation Rationale: Samples need to be dehydrated before being embedded in a resin. However, classic dehydration techniques will result in the loss of DMSP from the cells. To effectively prevent the loss of DMSP from the cells, the entire sampling procedure leading to NanoSIMS analysis must be water-free, with all steps performed under strict anhydrous conditions. For this, we use cryopreservation techniques followed by freeze substitution in an acrolein-ether mixt...