To estimate histological changes, embedded pancreatic tissues were sectioned to a thickness of 8 μm for hematoxylin/eosin (HE) staining (G1121; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), Masson’s trichrome staining (G1346; Beijing Solarbio Science s& Technology Co., Ltd., Beijing, China), and immunofluorescent staining. Mouse anti-insulin (rat, 1:500, ab181547; Abcam, Waltham, MA, USA), rabbit anti-glucagon (rabbit, 1:150, 15954-1-AP; Proteintech, Rosemont, IL, USA), and the corresponding secondary antibodies (donkey anti-rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555; donkey anti-mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488(REF.A21202); Life Technologies, Carlsbad, CA, USA) were used to detect alpha and beta islet cells. As described in Chansela et al.’s study, the islets could be categorized as small (<10,000 μm2), medium (10,000–50,000 μm2), large (50,000–100,000 μm2), and extra large (>100,000 μm2) on the basis of the total area of islets [43 (link)]. The islet density is calculated as follows: the number of each islet size/total number of islets ×100 [43 (link)].
G1121
Manufactured by Solarbio
Sourced in China
The G1121 is a laboratory equipment product. It serves a core function in laboratory settings.
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6 protocols using g1121
1
Histological Analysis of Pancreatic Islets
2
Immunohistochemical Analysis of KIAA1217
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An immunohistochemistry assay was performed to detect KIAA1217 expression in the TMA tissue samples. The TMA slide was dewaxed in xylene and rehydrated with a series of graded alcohol solutions. Then, antigen retrieval was performed with sodium citrate buffer (C1032, Solarbio, Beijing, China). According to the protocols of the two-step detection kit (PV-9001, ZSGB-BIO, Beijing, China), the TMA slide was incubated in an appropriate endogenous peroxidase blocker for 10 min at room temperature, followed by incubation with anti-SKT (KIAA1217 is also known as SKT) antibodies overnight at 4 °C and subsequent incubation with response enhancer and enhanced enzyme-labeled goat anti-rabbit IgG polymer for 20 min at room temperature. A DAB Chromogenic Kit (ZLI-9017, ZSGB-BIO, Beijing, China) was used to detect antibody binding, and the reaction was stopped by immersing the TMAs in running water once a brown color appeared. Finally, the TMA slide was counterstained with hematoxylin (G1121, Solarbio, Beijing, China), dehydrated using a series of graded alcohol solutions, and mounted. Images were photographed with an inverted microscope. Appropriate positive and negative controls were included for each run of the IHC assay. The antibodies used in this study are listed in Supplementary Table S3 .
3
Characterization of Decellularized Kidney ECM
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To characterize the quality of decellularization, the tissues before and after decellularization were fixed in 4% PFA for 30 min, immersed in 15% and then 30% sucrose solution, and embedded in OCT for cryosection. H&E staining was performed according to the manufacture protocol (Solarbio, Cat#G1121).
SDS‐PAGE was performed to qualitatively examine ECM protein content. Briefly, a small piece of native porcine kidney tissue was lysed in RIPA buffer. Digested ECM hydrogel was used because the decellularized ECM did not dissolve in common lysis buffers. The protein concentration of each sample was determined by BCA assay. Proteins were separated by 10% SDS‐PAGE, stained by Coomassie Blue, digested by trypsin and identified by LC‐MS/MS as previously described [31 (link)].
DNA quantification was performed by PicoGreen assay following previous reports [10 (link)]. Freeze‐dried porcine kidney tissues before and after decellularization were digested in Proteinase K overnight. DNA was quantified by the PicoGreen dsDNA Kit (Yeasen, Cat#12641ES01). Sulfated glycosaminoglycan (GAG) was quantified by the DMMB assay following the previous report [10 (link)].
SDS‐PAGE was performed to qualitatively examine ECM protein content. Briefly, a small piece of native porcine kidney tissue was lysed in RIPA buffer. Digested ECM hydrogel was used because the decellularized ECM did not dissolve in common lysis buffers. The protein concentration of each sample was determined by BCA assay. Proteins were separated by 10% SDS‐PAGE, stained by Coomassie Blue, digested by trypsin and identified by LC‐MS/MS as previously described [31 (link)].
DNA quantification was performed by PicoGreen assay following previous reports [10 (link)]. Freeze‐dried porcine kidney tissues before and after decellularization were digested in Proteinase K overnight. DNA was quantified by the PicoGreen dsDNA Kit (Yeasen, Cat#12641ES01). Sulfated glycosaminoglycan (GAG) was quantified by the DMMB assay following the previous report [10 (link)].
4
Histological Analysis of Rat Biceps
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Biceps brachii muscle tissues of rats were harvested from the rats and fixed with 4% paraformaldehyde (PFA) overnight. The tissues embedded in paraffin were sectioned into 4 μm thickness, followed by deparaffinization and rehydration, and finally stained with hematoxylin and eosin following the manufacturer's introductions (Solarbio, Beijing, G1121).
5
Quantitative Ovarian Follicle Analysis
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The paraffin-embedded ovaries were cut into serial sections and then one of every five sections was selected for HE-staining (Solarbio, G1121). Follicle count was done according to previous studies [32 (link), 33 (link)]. The criterion of the follicle count was done as follows, total number of follicles = normal follicles + atretic follicles; percentage of follicles at each stage = total number of follicles at each stage/total number of follicles.
6
Photothermal Therapy of Tumor-Bearing Mice
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Once the tumor sizes reached approximately 100 mm3, the mice were randomly assigned to five groups (each consisting of 5 mice) and subjected to the following treatments: (1) PBS + NIR; (2) Fin56 + NIR; (3) FSR-Fin56; and (4) FSR-Fin56 + NIR. At day 0, 4, 8, and 12, 150 µL of various treatment agents were injected into the tail vein of the tumor-bearing mice. The dose of Fin56 at each injection was 7 mg kg−1 and the pH of PBS was 7.4. At 12 hours post-injection, the tumor site underwent 808 nm NIR laser irradiation (1.0 W/cm2, 6 minutes). Throughout the therapy period, the tumor volume and body weight of the mice were monitored every alternate day. After 16 days of treatment, blood samples were collected, and the tumors along with major organs (heart, liver, lung, spleen, and kidney) were fixed in 4% paraformaldehyde for subsequent experiments. Serum samples were obtained by centrifugation for hepatic and renal function analyses. The main organs and tumor tissues were sectioned for H&E (Solarbio, G1121) and immunofluorescence staining. Antibodies utilized included Ki67 (Affinity, AF0198, 1:200) and GPX4 (Huabio, ET1706-45, 1:100). Fluorescence-conjugated secondary antibodies included donkey anti-rabbit IgG (H + L) FITC (Alexa Fluor 488) (Invitrogen, A-212206; 1:500) and donkey anti-rabbit IgG (H + L) TRITC (Invitrogen, A16026; 1:500).
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