Formaldehyde solution

Fresh tissue was fixed in 4% formaldehyde solution (VWR) at room temperature, and embedded in paraffin and sectioned at 4 µm. HFO were fixed in 4% formaldehyde solution (VWR) for 1 h at 4°C and embedded in HistoGel (Thermo Fisher Scientific) prior to paraffin embedding and sectioned at 4 µm. Paraffin slides were deparaffinized and rehydrated. For antigen retrieval, slides were treated at 96°C for 10 min in 0.01 M sodium citrate buffer pH 6.0. Sections were blocked with PBT (PBS, 0.1% Triton X-100, 1% w/v BSA) and incubated overnight at 4°C with primary antibody anti-ARHGAP18 (ab175970, Abcam; 1:100) or anti-p-SQSTM1 (5 µg/ml; MBL). Tissue sections were stained at room temperature with fluorescent-labelled secondary antibody (goat anti-rabbit Alexa Fluor 488, A11008, lot 1966932, and goat anti-rat Alexa Fluor 546, A11081, lot 1661229; both Invitrogen; 1:500). Slides were mounted with ProLong Gold Antifade reagent with DAPI (Thermo Fisher Scientific). Images were taken with a Leica DM6000 microscope, using a 10× objective and 7× ocular and LAS AF software. Scoring of the immunohistochemistry slides was performed blinded for genotype. Specimen collection of primary colonic tissue was approved by the biobank review committee of the Academic Medical Center Amsterdam (number 178#A201470).

Morphology and adhesion of the coating were investigated
using SEM
(Leo Ultra 55, Carl Zeiss AG, Germany) at 2 kV accelerating voltage.
Cross-sections of the as-prepared coated substrates were sliced to
1 mm thickness with a microtome blade, samples mounted on sample stages
and sputter coated with gold for 1 min at 10 mA for enhanced imaging.
Additionally, SEM was used to study the particle coatings after
exposure to bacteria to understand bacteria–particle interaction
and observe any change in bacteria morphology. Coating samples were
first rehydrated in phosphate buffered saline (PBS) followed by incubation
in 10⁸ colony forming units per mililiter (CFU/mL) S. epidermidis suspension in 10% v/v TSB overnight. After
18 h, samples were washed three times with 1 mL of PBS. Bacteria were
fixed in 4% formaldehyde solution (VWR International AB, Sweden) for
2 h at room temperature. The samples were then subjected to a dehydration
procedure in an ethanol solution gradient (20%, 50%, 70%, 85%, and
99.5%) for 15 min per step, followed by an immersion in 50% v/v hexamethyldisilazane
(HDMS) solution in ethanol for 30 min and a final step in 100% HDMS.
Samples were placed in a fume hood until fully air-dried and sputter
coated with gold prior the SEM analysis.

After micro-CT imaging, all hind limbs were collected for histological analysis and joints were fixed in 4% formaldehyde solution (Klinipath BV, Netherlands) at room temperature for 1 week. Thereafter, joints were decalcified at room temperature in 0.5 M EDTA (VWR international BV) solution for a total of 8 weeks, re-fixating for 3 days in 4% formaldehyde solution every 2 weeks, and embedded in paraffin. Five micrometer transversal knee joint sections were cut and stained either with Safranin-O/Fast green or hematoxylin/eosin to evaluate cartilage degeneration using the Mankin score (Mankin & Lippiello, 1970 (link)) (0 complete healthy – 14 total joint destruction) or synovitis using the Krenn score (Krenn et al., 2006 (link)) (0 healthy – 9 severe synovitis), respectively. Scoring was done at random by two blinded observers independently and scores were averaged (AT and IR).

For histological analysis, tissue sections of the distal ileum and distal colon were fixed in 4% formaldehyde solution (VWR, Radnor, Pennsylvania, USA) and embedded in paraffin. 5 µm paraffin-embedded sections were stained with hematoxylin and eosin (Sigma-Aldrich). Intestinal inflammation was scored blindly by two independent observers using a validated scoring system, as previously described [32] (link).

Cell attachment was
assessed at day 1 and proliferation after days
7 and 11 by measuring the DNA content using Picogreen dye (Thermo
Fisher Scientific, USA) as described earlier.^{43 (link)} Briefly, 0.3 mL of lysate solution was prepared using 0.2 mg mL^–1 proteinase-k (Sigma-Aldrich, USA) and 0.02% sodium
dodecyl sulfate (SDS), added to the scaffolds, and incubated at 37
°C for 12 h. Further, lysate solution was collected, freeze–thawed
at −80 °C, and transferred to a 96-well TCP, and an equal
amount of Picogreen dye was added to each well and incubated for 5
min. Fluorescence measurements were recorded at 485 excitations and
520 emission wavelengths in a microplate reader. The dsDNA amount
was calculated using a standard curve plotted by a serial dilution
of a known DNA concentration.
For SEM, cells were fixed on the
scaffolds using a 4% formaldehyde solution (VWR, Sweden) for 30 min,
and scaffolds were then critically dried using a series of alcohols:
30, 50, 70, 90, and 100%. Confocal microscopy (Zeiss LSM 800) was
used to visualize cells in 3D scaffolds. Cells were fixed using 4%
formaldehyde, washed with PBS, and stained with fluorescent dyes.
Nuclei were stained with SYTOX Green (Thermo Fisher Scientific, USA)
in a 1:30000 dilution of stock solution and incubated for 15 min at
RT. The cell cytoskeleton was stained using Alexa Fluor 546 dye (6.6
μM; 30 min; Thermo Fisher Scientific, USA).

Cells were cultured onto glass coverslips for 24 h prior to fixating for the colocalization studies. Cells were fixed using 4% formaldehyde solution (VWR, Amsterdam, The Netherlands) and permeabilized in 0.05% Triton X-100 in PBS (Bio-Rad, Veenendaal, The Netherlands). Slides were blocked with 0.5% BSA in PBS and incubated overnight at 4°C with anti-ARHGAP18 (ab175970, Abcam; 1:100) and anti-p-SQSTM1 (Ser403, D344-3MS, MBL; 1:500). The slides were stained at room temperature with fluorescent-labelled secondary antibody (donkey-anti-rat AF488, A21208, lot 1476598, Life Technologies; donkey-anti-rabbit AF546, A10040, lot 1833519, Invitrogen; 1:200). Slides were mounted with ProLong Gold Antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific). Images were taken with a Leica DM6000 microscope using a 63× objective and 10× ocular, and LAS AF software (Leica).

Precirol ATO5 (glyceryl palmitostearate) was obtained from Gattefosse (Saint-Priest, France) and Miglyol 812 was received from Sasol (Hamburg, Germany). Carbopol 940 was purchased from Acros (Morris County, NJ, USA). Triethanolamine (TEA) was purchased from Merck (Darmstadt, Germany). RhTM protein and anti-rhTM antibody were provided by Blue Blood Biotech Corporation (Taipei, Taiwan). H&E stain, Poloxamer 188 (10% solution), bovine serum albumin (BSA) and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Streptozotocin (STZ) was obtained from Cayman Chemical (Ann Arbor, MI, USA). DMEM and fetal bovine serum (FBS) were purchased from GE Healthcare (Pittsburgh, PA, USA). Formaldehyde solution was from Avantor (Center Valley, PA, USA), and penicillin-streptomycin was from Gibco (Waltham, MA, USA).