Mar 16, 2026
protocols of DeepOrganelle
- Li Xiao1,
- Liqing Liu2,
- Hongjun Wu1,
- Jiayi Zhong3,
- Xixia Li2,
- Yan Zhang2,
- Junjie Hu2,
- Fei Sun2,
- Ge Yang2,
- Tao Xu2
- 1Beijing University of Posts and Telecommunications;
- 2Chinese Academy of Sciences;
- 3University of Chinese Academy of Sciences
- DeepOrganelle
Protocol Citation: Li Xiao, Liqing Liu, Hongjun Wu, Jiayi Zhong, Xixia Li, Yan Zhang, Junjie Hu, Fei Sun, Ge Yang, Tao Xu 2026. protocols of DeepOrganelle. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwwq32vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 15, 2026
Last Modified: March 16, 2026
Protocol Integer ID: 313286
Keywords: deeporganelle protocol, deeporganelle
Abstract
protocols of DeepOrganelle
Guidelines
**Ultra-thin Sectioning**
Resin samples were cut into ultra-thin sections 70-nm-thick using a microtome (EM UC7; Leica) and collected with silicon substrates to enhance the transductivity during scanning electron microscopy.
**Scanning Field Emission Electron Microscopy Data**
For large-scale tissue electron microscopy, tile sets covering one intact seminiferous tubule at 10-nm pixel resolution were acquired and stitched with Maps software using Helios 5 Hydra CX scanning microscopy (Thermo Fisher) with Circular Backscattered Detector under immersion high-magnification mode (4 mm working distance, 2 kV accelerating voltage, 40 nA beam current, 2 μs dwell time).
**FIB-SEM data collection**
3 dimensional Volume Electron Microscopic data were acquired on the Helios 5 Hydra CX system (Thermo Fisher) using Auto Slice and View acquisition software with O as plasma FIB ion source at 30kV, 5.6nA beam current, and with TLD detector using back-scattered electron signal under immersion high-magnification mode (4 mm working distance, 10 nm pixel resolution, 2kV accelerating voltage, 0.8 nA beam current, 2 μs dwell time).
Troubleshooting
Resin Sample Preparation
Expose the testes in the lower abdomen after anesthetizing the mouse via isoflurane inhalation.
Transpierce the pair of testes with a 26-gauge needle in the middle shaft of the short axis and inject slowly with 0.2 ml first fixative (2% PFA + 2.5% glutaraldehyde in PBS) from one end of the long axis for brief fixation of the seminiferous tissues under physiological conditions.
Dissect the testes and immerse into fresh first fixative.
Carefully remove the tunica albuginea using ophthalmic scissors, and gently dissect bundles of seminiferous tissue with approximately 20 seminiferous tubules using the ophthalmic scalpel.
Fix the tissue in first fixative for more than 12 h in a 4°C refrigerator.
Adapt reduced osmium-thiocarbohydrazide-osmium (ROTO) procedures for preparation of the seminiferous tissue sample.
Immerse dissected tissue in 2% (wt/vol) OsO4 in water at 4°C for 90 min, followed by a wash with 1.5% potassium ferrocyanide (wt/vol) at 4°C for 90 min.
Incubate tissues in filtered 1% thiocarbohydrazide at room temperature for 30 min, 2% unbuffered OsO4 aqueous solution at 4°C for 90 min, and 2% uranyl acetate aqueous solution at 4°C overnight followed by heating at 50°C for 60 min.
Perform triple rinses in ddH2O for 30 min between steps.
Dehydrate the tissues through a graded ethanol series (30%, 50%, 70%, 80%, 90%, 100%, 10 min each) at 4°C three times, followed by two exchanges of 100% acetone.
Infiltrate dehydrated samples in graded mixtures (3:1, 1:1, and 1:3) of acetone with SPI-PON812 resin, then pure resin for 2 days with exchange every 12 h.
Embed tissues in pure resin with 1.5% BDMA accelerator and align longitudinally with the wells of an embedding mold and polymerize in an oven at 45°C for 24 h and 60°C for 48 h.
Ultra-thin Sectioning
Cut resin samples into ultra-thin sections 70-nm-thick using a microtome (EM UC7; Leica) and collect with silicon substrates to enhance the transductivity during scanning electron microscopy.
Scanning Field Emission Electron Microscopy Data
Acquire tile sets covering one intact seminiferous tubule at 10-nm pixel resolution and stitch with Maps software using Helios 5 Hydra CX scanning microscopy (Thermo Fisher) with Circular Backscattered Detector under immersion high-magnification mode (4 mm working distance, 2 kV accelerating voltage, 40 nA beam current, 2 μs dwell time).
FIB-SEM data collection
Acquire 3-dimensional Volume Electron Microscopic data on the Helios 5 Hydra CX system (Thermo Fisher) using Auto Slice and View acquisition software with O as plasma FIB ion source at 30kV, 5.6nA beam current, and with TLD detector using back-scattered electron signal under immersion high-magnification mode (4 mm working distance, 10 nm pixel resolution, 2kV accelerating voltage, 0.8 nA beam current, 2 μs dwell time).