A microscope

MES23.5 cells were seeded onto coverslips that were pre-coated with poly-L-lysine at room temperature. Then, the cells were fixed with 4% paraformaldehyde for 10 min and then permeabilized with 1% Triton X-100 in PBS, after which nonspecific binding was blocked with 1% bovine serum albumin (BSA). Then, the cells were incubated with a primary antibody against TUBB3 (1:50, Sigma) in 1% BSA overnight at 4° C, which followed by an incubation with an Alexa-488 conjugated secondary antibody (1:500, Invitrogen) at room temperature for 1 h. The coverslips then were washed three times with PBS, and the samples were imaged under a microscope (Zeiss, Germany).

To test Ki‐67 level, tissue samples were stained. Primary monoclonal probes for Ki‐67 were used for tumor detection at 4°C overnight. After that, a suitable second antibody was used for incubation. Tissues underwent diaminobenzidine treatment and hematoxylin counterstaining. A microscope (Carl Zeiss) was applied for tissue observation.

The tumor cell culture medium was changed to serum-free RPMI-1640 or DMEM medium for 48 h and then collected, centrifuged and filtered to obtain tumor conditioned medium (CM). The wells of the 96-well plate were coated with 50µl pre-thawed Matrigel (BD Biosciences, USA) and incubated for 1 h in a 37 ℃ incubator. 1 x 10⁴ HUVECs were seeded on the gel with 200µl of CM concentrated using an ultrafiltration device (Millipore, USA). The tube formation of HUVECs was observed after incubated 12 h using a microscope (Zeiss, Germany).

After general anesthesia, according to the individual conditions of the patient, blood pressure and partial pressure of carbon dioxide were controlled [18 (link),19 (link),20 ]. The extended pterional approach was used; the temporal muscle and STA were carefully separated, the bone flap was removed, and the dura mater was carefully cut to expose the brain surface. A microscope (ZEISS) was used to perform a fluorescence contrast test to understand the blood flow of the distal middle cerebral artery on the brain surface and select the appropriate blood vessels. The recipient blood vessel and STA branch were wrapped and soaked with papaverine solution to prevent vasospasm. Subsequently, a temporary aneurysm clip was used to clamp the blood supply on both sides of the recipient blood vessel and proximal end of the STA branch. The frontal parietal surface was examined at the distal end of the body vessel and the STA branch. After bypass was completed, intraoperative angiography was performed to check the patency and flow of the reconstructed vessel. Finally, the separated and intact temporal muscles were directly applied to the parietal and frontal brain surfaces, and the dura mater was turned over and applied to the brain surface to complete the combined cerebral revascularization operation [21 (link),22 (link)].

Sterilize the Fertile eggs with 70% ethanol and put them in 37 °C, 45 ± 5% Humidity incubator. After 72 h incubation, drill a hole in the air sac of the Fertile egg and check the vessel. Then, put a disk containing TJFs (0.1 and 1%) and Vitamin C (0.1%) with sterilized silicon ring in the Chicken Chorioallantoic Membrane. block it with a tegarderm (sterile barrier to external contaminants) and put it back into 37 °C, 45 ± 5% Humidity incubator. After 72 h, Verify the angiogenensis effects of the TJFs through a microscope (Carl Zeiss, USA).

Paraffin-fixed sagittal brain sections were processed for Aβ immunohistochemistry. Sections were boiled in citrate buffer (pH 6.0, 10 mM trisodium citrate) for 5 min followed by cooling. After blocking with 5% goat serum (S-1000, Vector Laboratories Inc) for 30 min, brain sections were incubated with an anti-82E1 (1 : 100, 10323, IBL) antibody overnight at 4°C followed by incubation with goat anti-mouse Alexa Fluor 488 (A11029, Thermo Fisher Scientific) for 1 h at room temperature. Nuclei were stained with DAPI (P36931, Vector Laboratories Inc). Images were obtained using a microscope (Carl Zeiss). Aβ plaques were evaluated as the percentage of the immunostained area (positive pixels) divided by the total area examined (total pixels) using ImageJ software.