TITLE: Sectioning and HE staining of Mouse Brain and embryo by Cryostat
AUTHORS: Yuting Fu, jiashikai, Xiaodong Liu

[DESCRIPTION]
This protocol describes how to use the cryostat to prepare and slice mouse brain and embryo sections for HE staining and following spatial transcriptomic experiments.

[STEPS]
SECTION: Preparation Methods for E12.5 mouse-embryo-eye
5. Mouse embryos were collected from pregnant C57BL/6J female mice at embryonic day 12.5
(E12.5). Mouse brain was dissected from 8-week-old C57BL/6J male mice;
SECTION: Preparation Methods for E12.5 mouse-embryo-eye
1. E12.5 pregnant female mice was anesthetized with carbon dioxide, the whole uterus was
collected and washed 3 times in ice-cold DPBS;

SECTION: Preparation Methods for E12.5 mouse-embryo-eye
2. Uterus was separated under stereo microscope, each embryo was numbered and
photographed with Motorized Fluorescence Stereo Zoom microscope (ZEISS, Axio
Zoom V16); 
  

SECTION: Preparation Methods for E12.5 mouse-embryo-eye
3. The yolk sac was collected to extracted DNA for genotyping (identification of sex);

SECTION: Preparation Methods for E12.5 mouse-embryo-eye
4. Using dust-free paper to gently wiped the liquid on the surface of the embryo, the
embryo was rinsed with ice-cold Tissue-Tek OCT (Sakura, 4583), and then moved
to encapsulation box with ice-cold OCT;

SECTION: Preparation Methods for E12.5 mouse-embryo-eye
6. The air bubbles were carefully removed with the syringe, and the embryo was placed
in sagittal position with tweezers;     
   


SECTION: Preparation Methods for E12.5 mouse-embryo-eye
7. The location of embryonic eye was circled, and marked the orientation of embryo,
then tissues was transferred to a -80℃ freezer for snap-frozen and storage;

SECTION: Preparation Methods for E12.5 mouse-embryo-eye
8. Embryos of average size and normal phenotype were selected for subsequent cryosection
and sequencing (note: the embryos used in our benchmarking analysis came from a
litter of mice);

SECTION: Preparation Methods for E12.5 mouse-embryo-eye
9. Before sectioning, tissue block was took out from -80℃ freezer and placed in cryostat (Leica, CM1950) to balance for at least 30 min;  


SECTION: Preparation Methods for E12.5 mouse-embryo-eye
10. The tissue block was smoothly glued to the sample head, so that the embryo was
sectioned in sagittal position. If necessary, the angle can be fine-tuned so
that the blade section is strictly parallel to the cross-section of the tissue
block;

SECTION: Preparation Methods for E12.5 mouse-embryo-eye
11. Cryosections were cut at a thickness of 10 μm, both left eye and right eye can be collected;

SECTION: Preparation Methods for E12.5 mouse-embryo-eye
12. The structure of the sequenced cryosections were shown in the followed image:
      

SECTION: Preparation Methods for E12.5 mouse-embryo-eye
13. HE staining procedure: cryosections were balanced at room temperature for 30 min,
and then fixed with 4% PFA for 3 min. Then, the sections were washed with ddH2O
for 2 min, stained with hematoxylin for 6 min, washed with ddH2O,
stained with eosin for 2 min, washed with ddH2O. After that, sections were gradient dehydrated (75% ethyl alcohol for 1 s, 85% ethyl alcohol for 1 s, 95% ethyl alcohol for 1 s, 100% ethyl alcohol for 1 s, 100% ethyl alcohol for 1 min), cleared (xylene for twice), and sealed with Permount TM Mounting Medium after airing. Finally, the figure was scanned using Motorized Fluorescence Microscope (Nikon, Ni-E).

SECTION: Preparation Methods for male mouse brain
14. 8-week-old male mice was anesthetized with carbon dioxide and decapitated;

SECTION: Preparation Methods for male mouse brain
15. The whole brain was rapidly dissected, numbered and photographed with Motorized
Fluorescence Stereo Zoom microscope;
  

SECTION: Preparation Methods for male mouse brain
16. Using dust-free paper to gently wiped the liquid on the surface of the brain, the
brain was rinsed with ice-cold Tissue-Tek OCT (Sakura, 4583), and then moved to
encapsulation box with ice-cold OCT;
    

SECTION: Preparation Methods for male mouse brain
17. The air bubbles were carefully removed with the syringe, and the brain was placed
properly with tweezers;

SECTION: Preparation Methods for male mouse brain
18. The location of hippocampus was circled, and marked the orientation of brain, then
tissues was transferred to a -80℃ freezer for snap-frozen and storage;

SECTION: Preparation Methods for male mouse brain
19. Brain samples of average size and normal phenotype were selected for subsequent cryosection
and sequencing;

SECTION: Preparation Methods for male mouse brain
20. Before sectioning, tissue block was took out from -80℃ freezer and placed in cryostat (Leica, CM1950) to balance for at least 1 h;
 
  

SECTION: Preparation Methods for male mouse brain
21. The tissue block was smoothly glued to the sample head, and the cerebellum was oriented towards the experimenter, so that the brain was sectioned in coronal position. If necessary, the angle can be fine-tuned so that the blade section is strictly parallel to the cross-section of the tissue block;

SECTION: Preparation Methods for male mouse brain
22. Cryosections were cut at a thickness of 10 μm;

SECTION: Preparation Methods for male mouse brain
23. The structure of the sequenced cryosections were shown in the followed image:
      HE staining of cryosections. The distance of cryosections was marked.

SECTION: Preparation Methods for male mouse brain
24. HE staining procedure: cryosections were balanced at room temperature for 30 min,
and then fixed with 4% PFA for 3 min. Then, the sections were washed with ddH2O
for 2 min, stained with hematoxylin for 6 min, washed with ddH2O, stained with eosin for 1 min, washed with ddH2O. After that, sections were gradient dehydrated (75% ethyl alcohol for 1 s, 85% ethyl alcohol for 1 s, 95% ethyl alcohol for 1 s, 100% ethyl alcohol for 1 s, 100% ethyl alcohol for 1 min), cleared (xylene for twice), and sealed after airing. Finally, the figure was scanned using Motorized Fluorescence Microscope (Nikon, Ni-E).