Phosphate buffered saline (pbs)

The rats were anesthetized with chloral hydrate (400 mg/kg) and then perfused transcranial with saline followed by 4% paraformaldehyde (PFA) (pH 7.4). For immunohistochemical labeling, rat brain tissues were fixed in 4% PFA and embedded in paraffin. Brain sections (5 μm thick) were prepared, deparaffinized and rehydrated in gradient ethanol. The slides were blocked with 10% goat serum in Phosphate Buffered Saline (PBS) (Abcam, Cambridge), and then incubated with the following primary antibody mixtures: anti-TSPO (1:200, affinity), anti-Iba-1 (1:200, Abcam), anti-NLRP3 (1:200, affinity) at 4 °C overnight. After being washed three times with PBS, the slices were incubated with goat anti-rabbit secondary antibody, washed again with PBS three times, stained with DAB (3,3′-diaminoben-zidine tetrahydrochloride) (Abcam, Cambridge) and counterstained with hematoxylin, and finally mounted, dehydrated, and covered with picks. Finally, the number of positive cells in three visual fields randomly selected by two people under a 40x light microscope was counted, and the average value was calculated for comparison between groups.

One mmol of folic acid in 20 ml DMSO with 1 mmol
of ethyl dimethylaminopropyl carbodiimide (EDC) and 2
mmol of N-Hydroxysuccinimide (NHS) was dissolved in
with a magnetic stirrer for 12 hours at room temperature.
Then, to remove excess material, the solution was filtered
and added to the ethylenediamine (EDA) as a linker, and
pyridine (0.2/ml) and acetonitrile added to precipitate the
folate. The precipitate was washed 3 times with diethyl
ether and then dried to give a yellow deposit. 10 mg of
MiR-PLGA was dissolved in 5 ml phosphate-buffered
saline (PBS, Abcam, UK) at pH=7.4 and sonicated
for 1 minute. 1 mg/ml EDC and 1 mg/ml of NHS were
dissolved in DW and each of them washed, and about
500 μl was added to the above solution and agitated with
a magenetic stirrer at room temperature for 2 hours and
then centrifuged for 20 minutes to remove EDC and NHS.
Following, folic acid was added to the product with the
ratio of 1 mg/ml in PBS, incubated overnight at room
temperature, agitated with the magenetic stirrer and then
centrifuged. Using the same procedure as a previous
study (17 (link)) the obtained solution was mixeded at room
temperature once more and then the extra non-conjugated
material was picked up by ultracentrifugation. Finally, the
residual solution was lyophilized and stored. Conjugation
of folate on the PLGA surface was confirmed by fourier
transform infrared spectroscopy (FTIR) analysis.

After sample preparation, the 39 sections were washed twice in PBS (Sigma-Aldrich) for 5 min. The sections were blocked with a solution of 10% normal serum (FBS; Gibco, Grand Island, NY) and 1% BSA (Sigma-Aldrich) in PBS for 3 h and then incubated with anti-β2M antibody (ab195531, Abcam, Cambridge, MA) at 1:400 dilution for 30 min. We added 3,3′-diaminobenzidine substrate (Abcam) and incubated the sectioned samples for 5 min, followed by thorough washing with tap water for another 5 min, and finally staining with Mayer’s Hematoxylin Solution for 10 min. The sections were dehydrated with a series of graded alcohol washes and then mounted for evaluation under light microscopy.

Paraffin embedded tissue sections (10 μm) were deparaffinized with xylene and rehydrated using a decreasing gradient of ethanol. Heat-induced antigen retrieval was performed using citrate buffer (10 mM Citric acid, 0.05% Tween-20, pH 6.0) for 20 min at 90–95°C. To reduce non-specific binding, blocking was carried out with 10% Normal donkey serum (EMD Millipore, Burlington, Massachusetts) and 0.2% Triton-X100 in PBS for 1 h at room temperature. A list of all the primary and secondary antibodies used are listed in Supplementary Table 3. Primary antibodies were prepared in antibody diluent solution (2.5% Normal donkey serum, 0.25% sodium azide, 0.2% trition X 100, and PBS, Abcam, Cambridge, Massachusetts), and sections were covered with primary antibodies overnight at 4°C. Next, each well was washed with PBS five times and then incubated with the respective secondary antibodies for 1 h, followed by mounting with VECTASHIELD® antifade mounting medium containing DAPI (Vector Labs, Burlingame, California) on glass sides. Slides were dried overnight and then imaged using a Nikon Eclipse C1 microscope.

An intraperitoneal injection of an aqueous solution of BrdU (120 mg/kg) at 90 min before death, in addition to 10 μL of a 10 μM solution of BrdU spotted on the wound site was performed as adapted from Mysorekar and Hultgren [42 (link)]. After sacrifice, excised wounds were placed in DMEM (Gibco) medium containing 500 μg/ml of gentamicin and penicillin G, andtreated with 0.5 mg/ml Liberase TL (Roche) for 1 h to create a single cell suspension. Cells were then fixed in 4% paraformaldehyde (PFA), solubilized in 2 N HCl solution at 37°C for 30 min, washed three times with PBS (Gibco) and further stained with rat α-CD45 (Abcam, Cambridge, UK) antibody for 1 h followed by three times PBS washes and addition of secondary antibody α-rat-Alexa Fluor 568 (Abcam, Cambridge, UK) for 1h. Secondary antibody was then washed off with PBS three times and samples were incubated with a rat α-BrdU-Alexa Fluor 647 (Abcam, Cambridge, UK) conjugate was added overnight in 0.1% Triton X–100 as per α-BrdU antibody manufacturer instructions. After the α-BrdU antibody was washed off with PBS three times, samples were incubated with a 1:1000 dilution of Hoechst 33342 for 5 min at room temperature with subsequent PBS washing three times. Cells were mounted with ProLong Glass Antifade Mountant (Invitrogen, Thermo Fisher Scientific, Singapore).

The expression of TfR on the surface of HepG2 cells and HL-7702 cells were examined by confocal laser scanning microscope (CLSM) and flow cytometry. CLSM images were obtained by fixation of HepG2 and HL-7702 cells using paraformaldehyde (4%) for 15 min at room temperature. Cells were then incubated with 5% bovine serum albumin (BSA) and 0.1% Tween for 1 h, followed by incubation with rabbit anti-TfR antibody (1/500, v/v, in 0.01 M PBS, Abcam, MA, USA) at 4^oC overnight. Cells were incubated with Alexa594-conjugated mouse anti-rabbit IgG (1/1000, v/v, in 0.01 M PBS Solarbio, China) for 1 h in darkness, and then stained with 100 µg/mL DAPI in darkness. The control group was incubated with 5% BSA to eliminate the nonspecific binding.
For flow cytometry, HepG2 and HL-7702 cells seeded in 6-well plates were suspended in PBS (at the concentration of 1×10⁶) and incubated with PE-conjugated anti-TfR (3 µL) for 30 min in darkness. Flow cytometry (FACS Calibur, BD Biosciences; San Jose, CA, USA) was performed to determine the expression of TfR on the surface of HepG2 cells and HL-7702 cells.