Supplies and reagents
All experiments had been carried out utilizing high-purity reagents and supplies. Ferric hexacyanoferrate and Cerium(III) nitrate hexahydrate had been obtained from Sigma-Aldrich (St. Louis, MO, USA). Tween 20, acetic acid (HAc), sodium acetate (NaAc), and paraformaldehyde (PFA) had been procured from Sinopharm Chemical Reagents Co., Ltd. (Shanghai, China). Hydrogen peroxide (H₂O₂, 30% w/w in H₂O) was bought from Merck KGaA (Darmstadt, Germany). Cyanine 5 NHS ester (Cy5) was obtained from Lumiprobe Company (Hunt Valley, MD, USA). The superoxide dismutase (SOD) exercise assay package (WST-8), cell counting kit-8 (CCK-8), Hoechst 33,342, propidium iodide (PI)/Calcein-AM twin staining package, and a pair of′,7′-dichlorofluorescin diacetate (DCFH-DA) had been sourced from Beyotime Institute of Biotechnology (Shanghai, China). The ELISA kits (Elabscience Biotechnology Co., Ltd., Wuhan, China) used had been: NLRP3 (E-EL-H2557), Caspase-1 (E-EL- M0201), GSDMD-NT (ab23627, Abcam), IL-18 (E-EL-M0730), IL-1β (E-EL-M0037), and Aβ (E-EL- M3010). For Western blot evaluation, the first antibodies used had been: ASC (ab283684, Abcam), Caspase-1 (ab207802, Abcam), GFAP (ab7260, Abcam), Iba-1 (ab178846, Abcam), Aβ (ab201060, Abcam), and GAPDH (ab8245, Abcam). Dulbecco’s Modified Eagle Medium/Nutrient Combination F-12 (DMEM/F12), fetal bovine serum (FBS), and SH-SY5Y neuroblastoma cells had been provided by Thermo Fisher Scientific (Waltham, MA, USA). Electron microscopy grade fixative was obtained from Electron Microscopy Sciences (Hatfield, PA, USA). Main antibodies towards ASC, Caspase-1, GFAP, Iba-1, Aβ, and Thioflavin T (Th-T), in addition to Integrin β2, Mac-1, and Lymphocyte function-associated antigen 1 (LFA-1) had been procured from Abcam (Cambridge, UK). Na⁺/Okay⁺-ATPase antibody and HRP-conjugated goat anti-rabbit secondary antibodies had been bought from Cell Signaling Know-how (Danvers, MA, USA). Ultrapure water (18.2 MΩ·cm) was obtained from a Milli-Q Benefit A10 water purification system (Merck Millipore, Burlington, MA, USA).
Instrumentation
Crystalline constructions had been analyzed utilizing powder X-ray diffraction (XRD) patterns obtained with a Rigaku Miniflex-600 diffractometer (Tokyo, Japan). Excessive-resolution morphological characterization was carried out utilizing a Hitachi H-7700 transmission electron microscope (TEM) (Tokyo, Japan). Elemental composition and distribution had been additional elucidated by way of energy-dispersive X-ray spectroscopy (EDS) mapping, carried out on a JEOL JEM-2100F field-emission TEM (Tokyo, Japan). Floor chemical states had been investigated by way of X-ray photoelectron spectroscopy (XPS) utilizing a Thermo Scientific Okay-Alpha + XPS system (Waltham, MA, USA). Colloidal properties, together with hydrodynamic diameter and zeta potential, had been assessed by way of dynamic gentle scattering (DLS) utilizing a Brookhaven NanoBrook Omni particle dimension analyzer (Holtsville, NY, USA).
Dissolved oxygen concentrations had been quantified utilizing a YSI 5100 dissolved oxygen meter (Yellow Springs, OH, USA). Fluorescence imaging was carried out on a Zeiss LSM 800 confocal laser scanning microscope (Oberkochen, Germany). Spectrophotometric measurements had been obtained utilizing a BioTek Synergy H1 microplate reader (Winooski, VT, USA). In vivo optical and X-ray imaging had been carried out utilizing a PerkinElmer IVIS Spectrum CT system (Waltham, MA, USA). Protein electrophoresis was carried out on a Bio-Rad Mini-PROTEAN Tetra vertical electrophoresis system, with subsequent imaging on a Bio-Rad ChemDoc XRS + imaging system (Hercules, CA, USA).
Pattern preparation concerned centrifugation steps utilizing an Eppendorf 5430R refrigerated microcentrifuge (Hamburg, Germany). Behavioral parameters had been recorded and analyzed utilizing EthoVision XT video monitoring software program from Noldus Data Know-how (Wageningen, Netherlands).
Synthesis of cerium-doped Prussian blue (PB-Ce) nanoparticles
The synthesis of cerium-doped Prussian Blue nanoparticles was carried out utilizing a modified hydrothermal technique. Briefly, 0.05 g potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) was dissolved in 30 mL ddH2O as An answer. Then 0.075 g cerium(III) nitrate hexahydrate (Ce(NO₃)₃·6H₂O) and 0.147 g sodium citrate trihydrate had been dissolved in 20 mL ddH2O as B resolution. Then A and B resolution had been combined in an ice bathtub and Stir for 30 min. The PB-Ce nanoparticles had been obtained by centrifugation and washed with ethyl alcohol for 3 occasions.
Lastly, the HPB-Ce nanoparticles had been redispersed in sterile phosphate-buffered saline (PBS, pH 7.4) and saved at 4 °C for additional use. The focus of the nanoparticle suspension was decided by lyophilizing an aliquot and measuring the dry weight.
Synthesis of neutrophil membrane-camouflaged PB-Ce nanoparticles (NM@PB-Ce)
The fabrication of neutrophil membrane-camouflaged PB-Ce nanoparticles (NM@PB-Ce) was achieved by way of a biomimetic coating course of using purified neutrophil cell membranes and synthesized PB-Ce nanoparticles.
Neutrophil membranes and PB-Ce nanoparticles had been mixed at a rigorously optimized protein-to-nanoparticle weight ratio. The combination was then subjected to a sequence of extrusion steps utilizing an Avanti mini-extruder (Avanti Polar Lipids, USA) outfitted with polycarbonate membranes of lowering pore sizes. Initially, the pattern was handed by way of a membrane with a pore dimension of 1 μm for 5 cycles, adopted by ten extrusion cycles by way of a 450 nm pore dimension membrane. This sequential extrusion course of ensures uniform coating and optimum dimension distribution of the ensuing nanoparticles. The extrusion parameters had been as follows:
Publish-extrusion, the NM@PB-Ce nanoparticles had been purified by differential centrifugation to take away unbound membrane fragments and extra PB-Ce cores. The pattern was centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was discarded, and the pellet containing the NM@PB-Ce nanoparticles was gently resuspended in sterile, endotoxin-free phosphate-buffered saline (PBS, pH 7.4). This washing course of was repeated 3 times to make sure excessive purity of the ultimate product.
The focus of the purified NM@PB-Ce nanoparticles was decided utilizing a mixture of bicinchoninic acid (BCA) protein assay for membrane quantification and inductively coupled plasma mass spectrometry (ICP-MS) for PB-Ce core quantification. The scale distribution and zeta potential of the nanoparticles had been characterised utilizing dynamic gentle scattering (DLS) and laser Doppler velocimetry, respectively.
The ensuing NM@PB-Ce nanoparticles had been saved in light-protected vials at 4 °C for short-term use (as much as 1 week) or lyophilized within the presence of 5% (w/v) trehalose as a cryoprotectant for long-term storage at – 80 °C.
Analysis of SOD-like exercise of NM@PB-Ce nanoparticles
The experimental process entails getting ready NM@PB-Ce nanoparticles in concentrations of 25, 50, 100, and 200 μg/mL within the assay buffer. In a 96-well microplate, 20 μL of every pattern or normal is combined with 200 μL of WST working resolution. After including 20 μL of enzyme working resolution to provoke the response, the plate is incubated at 37 °C for 30 min in a humidified setting. Absorbance at 450 nm is then measured utilizing a Synergy H1 microplate reader.
Evaluation of CAT-like exercise of NM@PB-Ce nanoparticles
NM@PB-Ce nanoparticles had been ready in a focus sequence of 0, 25, 50, 100, 150, and 200 μg/mL in phosphate-buffered saline (PBS, pH 7.4). A YSI 5100 dissolved oxygen meter outfitted with a Clark-type oxygen electrode was calibrated based on the producer’s directions. Then, 2 mL of every nanoparticle suspension was added to a sealed, temperature-controlled response chamber maintained at 25 ± 0.1 °C. The response was initiated by quickly injecting 20 μL of 30% H₂O₂ (last focus: 100 mM) into the chamber. Oxygen evolution was repeatedly monitored for five min, with information factors recorded each 5 s.
Analysis of POD-like exercise of NM@PB-Ce nanoparticles
Hydroxyl radicals had been generated by way of the Fenton response utilizing FeSO₄·7H₂O and H₂O₂. In a 96-well microplate, the next reagents had been added sequentially: 50 μL of FeSO₄·7H₂O (1.8 mM), 50 μL of nanoparticle suspension (PB or NM@PB-Ce) at varied concentrations (0–200 μg/mL), 50 μL of salicylic acid (3 mM), and 50 μL of H₂O₂ (0.03%). The response combination was then incubated at 37 °C for 30 min at nighttime. Absorbance was measured at 510 nm utilizing a Synergy H1 microplate reader to evaluate the outcomes.
Intracellular uptake and lysosomal colocalization of Cy5-labeled NM@PB-Ce nanozymes
The mobile internalization and subcellular localization of NM@PB-Ce nanozymes in SH-SY5Y neuroblastoma cells had been studied utilizing confocal microscopy. Cy5-labeled NM@PB-Ce nanozymes had been ready by conjugating with Cyanine 5 NHS ester, adopted by purification. SH-SY5Y cells had been incubated with these nanozymes at totally different time factors and lysosomes had been stained with LysoTracker Inexperienced, whereas cell nuclei had been counterstained with Hoechst 33,342. After washing, cells had been imaged utilizing a Zeiss LSM 800 confocal microscope, and Z-stack photos had been analyzed with ZEN Blue software program and ImageJ. Colocalization of nanozymes and lysosomes was assessed utilizing the JACoP plugin, calculating Manders’ overlap and Pearson’s correlation coefficients.
In vitro Alzheimer’s illness mannequin and experimental group design
An in vitro Alzheimer’s illness (AD) mannequin was established utilizing SH-SY5Y human neuroblastoma cells, incorporating genetic and environmental elements related to AD pathology. Amyloid-β (Aβ)₁₋₄₂ oligomers had been ready by dissolving the artificial peptide in HFIP, lyophilizing it, and resuspending in DMSO earlier than incubation to kind oligomers, which had been confirmed by transmission electron microscopy and Western blot. Aβ overexpressing SH-SY5Y cells had been handled with 50 μM oligomeric Aβ₁₋₄₂ for twenty-four h to induce AD-like pathology. 4 experimental teams had been arrange: management (wild-type cells), AD mannequin (Cells handled with Aβ₁₋₄₂), PB remedy (handled with Prussian Blue nanoparticles), and NM@PB-Ce remedy (handled with NM@PB-Ce). Cells had been cultured in DMEM/F12 with dietary supplements and maintained at 37 °C with 5% CO₂. The AD mannequin was validated by assessing mobile viability (CCK-8 assay), reactive oxygen species ranges (DCFH-DA assay), apoptosis charges (move cytometry), and AD-related protein expression (Western blot of Aβ and phosphorylated tau). Statistical evaluation was carried out with one-way ANOVA and Tukey’s post-hoc take a look at, contemplating p < 0.05 as important.
Cytotoxicity assessments in vitro
Cytotoxicity was evaluated utilizing the Cell Counting Package-8. SH-SY5Y cells had been plated in 96-well plates. Cells had been handled with NM@PB-Ce and PB throughout a focus gradient (0, 10, 20, 40, 80, 160 and 320 μg/mL) for twenty-four h. SH-SY5Y cells had been additionally uncovered to Aβ1–42 oligomers. (0, 20, 30 and 50 μM) to find out the optimum situation for inducing mobile stress mimicking illness phenotype. Primarily based on preliminary information, cells handled with 50 μM Aβ1–42 oligomers. Exhibited important phenotypic modifications according to AD and had been used for subsequent experiments. Publish-treatment, 10 μL of CCK-8 resolution was added to every properly, and the plates had been incubated for an extra 2 h at 37 °C in a humidified ambiance containing 5% CO2. Measuring absorbance at 450 nm with a SYNERGY H1 microplate reader. Following the CCK pointers. Mobile viability was calculated relative to untreated management cells, permitting for the evaluation of cytotoxic and protecting results of the remedies. Statistical evaluation was carried out utilizing one-way ANOVA with 6 unbiased organic replicates per group and three technical replicates per pattern. Publish-hoc comparisons had been carried out utilizing Tukey’s take a look at with α = 0.05. Mobile viability was calculated relative to untreated management cells, permitting for the evaluation of cytotoxic and protecting results of the remedies. Mobile toxicity in 4 teams cells was immediately visualized utilizing a stay/useless staining assay. SH-SY5Y cells had been cultured in confocal dishes, handled with PB and NM@PB-Ce Nanozyme for twenty-four h, and stained with calcein-AM and PI for 20 min. Fluorescence imaging was carried out utilizing CLSM.
In vitro reactive oxygen species (ROS) detection
The intracellular ROS ranges of the 4 teams cells had been quantified utilizing the two′,7′-dichlorofluorescin diacetate (DCFH-DA) fluorescent probe. Cells had been seeded in confocal dishes and incubated with DCFH-DA and Hoechst at a focus of 10 µM for 30 min at 37 °C at nighttime. Publish-incubation, cells had been washed with phosphate-buffered saline (PBS) to take away extra probe. Fluorescence depth, indicative of ROS manufacturing, was measured utilizing the CLSM at an excitation wavelength of 488 nm and emission wavelength of 525 nm.
In vitro protein expression evaluation
Protein ranges of Aβ, NLRP3, Caspase-1, GSDMD-NT, IL-18 and IL-1β had been analyzed by Western blotting. Cells from the 4 experimental teams (Management, AD, PB and NM@PB-Ce) had been harvested and lysed in RIPA buffer containing protease and phosphatase inhibitors. The protein focus was decided utilizing the BCA Protein Assay Package. Equal quantities of protein had been separated on SDS-PAGE gels and transferred onto PVDF membranes. The membranes had been blocked with 5% non-fat milk and incubated with major antibodies particular to Aβ, NLRP3, Caspase-1,GSDMD-NT,IL-18 and IL-1β in a single day at 4 °C. After washing, the membranes had been incubated with HRP-conjugated secondary antibodies. Protein bands had been visualized utilizing an enhanced chemiluminescence (ECL) detection system. Band depth was quantified with densitometry, normalized to GAPDH as a loading management.
Measurement of cell apoptosis by annexin V-FITC and PI staining
The evaluation of apoptosis induction by Aβ1–42 oligomers was carried out by way of Annexin V-FITC and PI staining. In accordance with the producer’s protocol, briefly, LNCaP and SH-SY5Y cells (1 × 105 cells/mL) had been handled with Aβ1–42 oligomers for twenty-four h. Subsequently, cells had been harvested, re-suspended within the binding buffer resolution, and stained with 5 µL of Annexin V-FITC and 10 µL of PI at room temperature for 15 min at nighttime. Then, 300 µL of Annexin-V binding buffer was added. The apoptotic cells had been decided utilizing FACSCantroII move cytometer and analyzed with FlowJo v.10.8.1 software program (FlowJo LLC, Ashland, OR, USA).
In vitro blood–mind barrier (BBB) transport assay
An in vitro BBB mannequin was established utilizing Transwell plates. B Finish.3 cells had been seeded within the higher compartment of the Transwell plate and cultured to kind a confluent monolayer, simulating the BBB. SH-SY5Y cells had been seeded within the decrease compartment to symbolize the mind parenchymal aspect. The tradition medium was refreshed each 2 days, and the integrity of the bEnd.3 cell monolayer was monitored by measuring the transepithelial electrical resistance (TEER) till a minimal worth of 300 Ω·cm2 was achieved, indicating the formation of a decent barrier. Cy5 labeled NM@PB-Ce was added to the higher chamber to judge its capacity to cross the BBB mannequin. The cells had been incubated with the compound for varied time factors (10, 30, 60, 90, and 120 min). Publish-incubation, the fluorescence depth of Cy5 throughout the higher and decrease compartment was quantified utilizing a PerkinElmer IVIS Lumina III system with an excitation wavelength of 670 nm to measure the transport effectivity of NM@PB-Ce throughout the BBB mannequin.
Animal grouping and remedy
This examine was accepted by the Ethics Committee of Fujian Medical College (approval quantity: 1ACUC FJMU2022-0608).
Sooner or later earlier than surgical procedure, animals had been acclimatized to the laboratory situations, and the spontaneous locomotor exercise was recorded; animals having regular spontaneous locomotion had been chosen for the surgical procedure. Every mice was anaesthetized with an intraperitoneal (i.p.) injection of 1.25% tribromoethanol (0.2 mL/mL). The 1.25% tribromoethanol was given to delay an aesthesia and analgesia in the course of the surgical procedure. The mice had been immobilized in a Stereotaxic body (Stoelting Co., USA, Mannequin no: 53311), and the dorsal head space was ready by trimming and shaving hairs. With the fontanel because the origin, 0.6 mm behind the fontanel and 1.1 mm to the left aspect, a small gap was made with a microdrill, and a 10-μl micro syringe was inserted to a depth of two mm, and a pair of.5 μL of Aβ1–42 oligomer was injected slowly with a micro syringe pump at a charge of 1 μL/min, and the tip of the needle was left in place for five min to permit full diffusion of the liquid after the injection. Lastly, the syringe was eliminated, the scalp was sutured, and the rats had been put again into the cage and waited for them to get up. The traditional management group was injected with an equal quantity of PBS resolution.
Management group: Mice obtained intraperitoneal injections of saline.
AD group: Mice obtained Intracranial stereotactic injection of Aβ1–42 oligomer.
PB group: AD mice obtained intravenous injections of PB (320 μg/mL, 0.01 mL/g/day) for 14 days.
NM@PB-Ce group: AD mice obtained intravenous injections of NM@PB-Ce Nanozyme (320 μg/mL, 0.01 mL/g/day) for 14 days.
Hemolysis assay
The hemolytic potential of NM@PB-Ce was assessed utilizing mouse blood. Freshly drawn blood was incubated with a sequence of NM@PB-Ce concentrations (0, 25, 50, 100, 200, 400, 800, and 1600 μg/mL) together with a management (H2O) to induce hemolysis. After incubation, the samples had been centrifuged, and the supernatant’s absorbance was measured at 540 nm to find out the diploma of hemolysis. The hemolysis charge was calculated by evaluating the absorbance of NM@PB-Ce handled samples to that of the management.
In vivo imaging and drug tissue distribution
In vivo imaging was carried out to judge the biodistribution of Cy5-labeled NM@PB-Ce. Management and AD mannequin mice had been administered with the compound by way of tail vein injection. Serial imaging was carried out at 5, 10, 20 30, 60, 120, 180, 240, and 300 min post-injection utilizing the PerkinElmer IVIS Lumina III system. Following the ultimate imaging level, mice had been euthanized, and main organs (coronary heart, liver, spleen, lungs, kidneys, and mind) had been harvested for ex vivo imaging. This allowed for the evaluation of NM@PB-Ce metabolism and distribution throughout the tissues.
Histological staining, hematological and biochemical evaluation
Following 14 days of drug intervention, mice had been euthanized, and tissues from the center, liver, spleen, lungs, kidneys, and mind areas (Hippocampus) had been harvested. Tissue sections had been ready and stained with hematoxylin and eosin (H&E) to judge morphological integrity and pathological modifications. Blood samples had been collected on the time of euthanasia for routine hematological and biochemical evaluation. Full blood depend (CBC) and blood haemato-biochemical had been carried out to evaluate the systemic impression of the drug intervention.
Behavioral experiments
Previous to group task and Aβ1–42 oligomer injection (Day 0), all animals underwent complete behavioral screening to make sure baseline equivalence. Open subject take a look at was carried out to evaluate common locomotor exercise and anxiety-like conduct. Moreover, swimming functionality was evaluated to make sure comparable motor operate earlier than water maze coaching. Solely animals exhibiting regular behavioral parameters had been chosen for the examine and randomly assigned to experimental teams.
Behavioral exams had been carried out on every group of mice after 14 days of drug intervention.
Open subject take a look at
The open subject take a look at was carried out to judge common locomotor exercise and anxiety-related conduct. Mice had been positioned in a sq. area, and their motion was tracked over a set interval (300 s). Parameters similar to distance traveled and time spent within the middle versus close to the partitions had been measured. Grid crossings had been quantified by counting the variety of occasions a mouse intersected the ground grid traces demarcated into equal-sized squares all through the open subject area. Every intersection was thought-about a ‘crossing’, and the whole variety of crossings was recorded during the take a look at. Grid crossing ranges had been categorized based mostly on the frequency of crossings, with particular thresholds set for low, medium, and excessive ranges of exercise. These thresholds had been decided empirically based mostly on the distribution of crossings throughout all take a look at topics.
Morris water maze
The Morris water maze was employed to evaluate spatial studying and reminiscence in mice. The experimental setup consisted of a round pool the place a platform was submerged just under the water floor. The pool was conceptually divided into 4 quadrants labeled in a clockwise sequence, with Quadrant 1 on the top-left and Quadrant 4 on the bottom-left, the place the platform was positioned. Over a 5-day acquisition part, mice had been skilled for 120 s per trial to find this hidden platform. Every trial was initiated from various beginning factors to make sure studying was not biased by the beginning place. Escape latencies, the time taken by the mice to search out and climb onto the submerged platform, had been recorded every day to gauge studying progress. On the ultimate day, a probe trial was carried out to judge spatial reminiscence retention, the place the platform was eliminated, and the time spent within the platform quadrant was recorded. To quantify spatial navigation effectivity, we calculated the ratio of the time every mouse spent heading immediately towards the platform’s location to the whole time of the trial. This ratio served as an index of navigational precision and reminiscence recall for the platform’s place. Moreover, enrichment scores had been derived to symbolize the proclivity of mice to discover particular quadrants. The scores mirrored the frequency at which totally different mouse teams visited every quadrant, offering a comparative measure of spatial desire, significantly noting the focused quadrant housing the submerged platform. This evaluation allowed us to deduce the retention of spatial reminiscence throughout varied remedy teams.
Y maze spontaneous alternation take a look at
The three arms of the labeled maze are A, B and C. Let the conduct software program efficiently join with the Y maze digital camera and alter the decision to completely seize the mice within the maze. Place the take a look at animal on the distal finish of the arm labeled A, dealing with the middle of the maze, and gently grasp the bottom of the tail. Depart the take a look at room. The mice had been allowed to discover the maze freely for 8 min. Mice had been faraway from the maze and returned to their authentic cages. Clear all partitions and flooring of the maze earlier than transferring on to the following animal. After completion of the experiment and testing of all designated mice, all mice had been returned to their dwelling cages within the holding room. Calculate the proportion alternation.
Western blot evaluation
Protein concentrations within the mind homogenates had been decided utilizing the Bradford assay. Equal quantities of protein had been loaded onto SDS-PAGE gels for separation and subsequently transferred onto PVDF membranes. The membranes had been blocked and incubated with major antibodies concentrating on Aβ, NLRP3, Caspase-1, GSDMD-NT, IL-18 and IL-1β. GAPDH was used as a loading management. Applicable HRP-conjugated secondary antibodies had been utilized, and the protein bands had been visualized utilizing enhanced chemiluminescence. Band intensities had been quantified utilizing densitometry.
Elisa evaluation
The degrees of NLRP3, Caspase-1, GSDMD-NT, IL-18, IL-1β, and Aβ had been quantified utilizing enzyme-linked immunosorbent assays (ELISA). The assays had been carried out based on the producer’s protocols. Briefly, the respective seize antibodies for NLRP3, Caspase-1, GSDMD-NT, IL-18, IL-1β, and Aβwere coated onto 96-well plates and incubated in a single day at 4 °C. After blocking with 5% BSA in PBS for 1 h, the supernatants or cell lysates had been added to the wells and incubated for two h at room temperature. Following in depth washing, the plates had been incubated with biotinylated detection antibodies for 1 h at room temperature, adopted by the addition of HRP-conjugated streptavidin for 30 min. After the ultimate wash, the substrate resolution was added to every properly, and the response was allowed to develop for 15 min. The absorbance was measured at 450 nm utilizing a microplate reader. The concentrations of the analytes had been decided utilizing normal curves generated with recombinant proteins.
Electron microscopy
For ultrastructural evaluation, striatal samples had been mounted, dehydrated, and embedded in resin. Ultrathin sections had been lower and stained with uranyl acetate and lead citrate earlier than examination underneath a transmission electron microscope.
Immunofluorescence for ACS, caspase-1, GFAP, Iba-1, Aβ and Th-T
Immunofluorescence staining was carried out on hippocampus sections to detect ACS, Caspase-1, GFAP, Iba-1, Aβ and Th-T. After blocking, sections had been incubated with major antibodies particular to every protein, adopted by fluorescently labeled secondary antibodies. Fluorescence was detected utilizing a confocal microscope, permitting for the localization and quantification of those proteins throughout the hippocampus tissue.
Golgi-cox staining
Golgi-Cox staining of the hippocampus tissue was carried out utilizing the FD Speedy GolgiStain™ Package (FD NeuroTechnologies, Hangzhou, China) following the producer’s protocol. Initially, all the mind was immersed within the staining resolution at room temperature for 14 days, shielded from gentle. Following this, the mind underwent a 3-day dehydration course of at 4 °C within the staining resolution. Coronal slices of the mind, every 100 μm thick, had been then ready utilizing a vibrating microtome (Leica, GER). These slices had been positioned in a chromogenic resolution and allowed to develop coloration for 10 min at room temperature. Photos of the PFC pyramidal neurons had been captured utilizing a microscope outfitted with a 100 × goal lens (Olympus BX61, JPN).
