Imagequant las 500

At 1, 7, and 14 days after SF hydrogel injection as well as 1 and 14 days after collagen hydrogel injection, the mouse brains were cut into coronal sections 1 mm thick and incubated with 4% paraformaldehyde (PFA) for 48 h. Images were acquired from both sides of each coronal section in a stereoscopic microscope (Leica, S6D, Wetzlar, Germany) coupled with a digital camera (MC170HD, Leica, Germany).
In mice implanted with SF-Ink or Collagen-Ink hydrogels, the amount of black matter for each slice of the brain was calculated by applying a color threshold to separate the gel from the surrounding tissue. The area occupied by the gel was calculated after the normalization of the image pixels with the corresponding scale and conversion of pixels into a SI mm² scaling system. The distribution of the hydrogel area across the rostrocaudal axis was based on classical anatomic brain references in mice [53 ]. For an estimation of hydrogel volume, the two faces of each brain section were considered as individual pieces with a thickness of 0.5 mm. In mice implanted with SF-Rho hydrogels, the size occupied by Silk-rhodamine fluorescence per brain section was captured in a fluorescence chamber via an ImageQuant™ LAS 500 (Cytiva, Marlborough, MA, USA) system with a constant fluorescent exposure time.

Harvested E. coli cells were treated with lysozyme, and whole-cell extracts were prepared by sonication. Total cell proteins were fractionated using Tricine-SDS-PAGE on 15% gels [51 (link)] and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-FL transfer membrane; Merck, Darmstadt, Germany). The proteins Rsd, RMF, YqjD, RpoA, and RplB were detected on the membranes using rabbit polyclonal antibodies against Rsd, RMF, YqjD, RpoA, and RplB, respectively. Immunostained membranes with ECL substrate (Cytiva, Tokyo, Japan) were scanned using ImageQuant LAS 500 (Cytiva, Tokyo, Japan). The density of each band on the membranes was quantified using ImageJ software (https://imagej.nih.gov/ij/index.html (accessed on 24 August 2022)). The linearity of the quantification was confirmed through several experiments with different amounts of sample solution loaded on an electrophoresis gel.

To observe the influence of HPH on exosomal marker protein expression, RAW-Exos treated with 10 cycles of HPH or non-treated RAW-Exos were enriched by ultracentrifugation (125,000× g, 70 min, 4 °C). Protein concentrations were then measured using a Micro BCA Protein Assay Kit. The resultant RAW-Exos (1 µg protein) were subjected to 10% SDS-PAGE and transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After blocking for 1 h at 37 °C with 3% bovine serum albumin (BSA) in Tris-HCl-buffered saline containing 0.1% Tween20 (pH 7.4), the PVDF membrane was reacted with anti-Alix mouse antibody (sc-53540; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-CD81 mouse antibody (sc-166029; Santa Cruz Biotechnology) at a dilution of 1:100 at 4 °C overnight, respectively. The membrane was then incubated with HRP-conjugated rabbit anti-mouse IgG (ab97046; Abcam, Cambridge, UK) at a dilution of 1:20,000 for 1 h at 37 °C. After reaction with Amersham ECL Prime Western Blotting Detection Reagent (Tokyo, Japan), protein bands were acquired with an ImageQuant LAS 500 (Cytiva). Band intensities were quantified using the image analysis software ImageJ (National Institutes of Health, Bethesda, MD, USA).

Total protein was extracted from inoculated leaves using protein extraction buffer (50 mM Tris-HCl, pH 6.8; 4.5% SDS; 7.5% 2-mercaptoethanol; 9 M Urea). The total proteins were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose (NC) membrane. The anti-PVX CP monoclonal antibody was used at a 1:5000 dilution and incubated with the membranes at 37 °C for 2 h. Subsequently, the membranes were soaked in 5% skim milk solution containing horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody. Finally, after rinsing in 1 x PBST several times, the membranes were used for the detection of chemiluminescence by using a Chemiluminescence imaging system (ImageQuant LAS 500, Cytiva, Marlborough, MA, USA).

A Nuclear and Cytoplasmic Extraction Reagents kit (78835, Thermo Fisher Scientific, USA) was used to extract cytoplasmic and nuclear proteins. A BCA Assay Kit (23223, Thermo Scientific, USA) was used to measure the protein concentration. In this experiment, the protein was electrophoresed on 10% SDS‒PAGE gels and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were coincubated overnight at 4 °C with specific primary antibodies (ab214429, ab32535, ab3778, ab41037, ab307674, ab32561, ab15580, ab133462, ab76429, ab124957, ab181602, ab32536, Abcam, Cambridge, UK) (PA5-61136, Invitrogen, USA) (18165-1-AP, Proteintech, China). The membranes were rewarmed for 2 hours and then incubated with rabbit IgG (ab97051, Abcam, Cambridge, UK) at 37 °C for 1 hour. The bands were detected using an ECL Western blot kit (32209, Thermo Fisher Scientific, USA) and ImageQuant™ LAS500 (Cytiva, China). In this study, GAPDH and Lamin B were used as controls. The methodology employed for the utilization of antibodies is documented in Supplementary Table 2.

The liver tissue was homogenized with a bead beater-type homogenizer in 10 volumes of 3 mM Tris-hydrogen chloride buffer (pH 7.4) containing 0.25 M sucrose, 1 mM ethylenediaminetetraacetic acid, and the protease inhibitor cocktail (Merck KGaA). After centrifugation (500 × g at 4°C for 10 min), the supernatant was used for western blotting analyses of CYP7A1 and GAPDH. First, the total protein content was determined using the protein assay BCA kit (Nacalai Tesque, Inc.). After the total protein (liver 5 mg of protein/lane) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (38 (link)), the separated proteins were transferred to a polyvinylidene fluoride membrane. Then, CYP7A1 and GAPDH expression levels were detected using a specific primary antibody (cat. no. sc-518007 and sc-32233; Santa Cruz Biotechnology Inc., Dellas, TX, USA), a horseradish peroxidase-conjugated secondary antibody (cat no. sc-516102, Santa Cruz Biotechnology Inc.), and chemiluminescent substrate solutions (ATTO Corporation, Tokyo, Japan), and detection of the band with ImageQuant LAS 500 (Cytiva, Tokyo, Japan) according to the manufacturer's instructions. The relative densities of each band were quantitatively determined using ImageQuant TL software (Cytiva) and normalized to GAPDH.