Neutral buffered formalin

Gadd45β-KO and C57BL/6 J mice (The Jackson Laboratory) were housed at a constant temperature (20–22 °C) on a 12:12-h light/dark schedule. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB-AEC-16165) and conducted in accordance with the committee’s guidelines. Ten-week-old male mice were used for the experiments. Acute colitis was induced by administering 3% or 5% (w/v) DSS in the drinking water. For the repair experiment, mice were acclimatized to 3% DSS for 5 days and then provided regular drinking water for 3 or 5 days. Weight changes were calculated as the percent change in weight compared with the baseline weight, and macroscopic scoring of colon tissue was estimated according to the following grading system: 0 = no inflammation, 1 = swelling or redness, 2 = swelling and redness, 3 = one or two ulcers, 4 = more than two ulcers or one large ulcer, 5 = mild necrosis, and 6 = severe necrosis. Colons were dissected and washed with phosphate-buffered saline (PBS). The distal colon was fixed in 10% neutral buffered formalin (BBC Biochemical, Mt. Vernon, WA, USA), and the other portion was frozen in liquid nitrogen (LN2) and stored at −80 °C.

MelanoDerm (MEL-300-B, MatTek Corp., Ashland, MA, USA), used for the skin-lightening study, was cultured in EPI-100-NMM-113-PRF medium (MatTek Corp., Ashland, MA, USA) at 37 °C in a humidified 5% CO₂ incubator. Different concentrations of pseudoalteromone A were added to the culture medium every other day for 14 days. Thereafter, epidermal pigmentation was examined by performing optical and histological analyses. The epidermal pigmentation level was calculated by comparing variations in L* values (a lightness/darkness index) on days 1 and 14 and estimating the difference between them (ΔL). For the histological examination, tissues were fixed in 10% neutral buffered formalin (BBC Biochemical, Mount Vernon, WA, USA) and embedded in paraffin. Paraffin-embedded samples were then sliced into 5 μm sections, stained with hematoxylin and eosin (H and E) and Fontana Masson’s (F-M) staining (for melanin), and then examined histologically.

Organs containing metastatic deposits showing luciferase expression were harvested and stabilized in 10% Neutral Buffered Formalin (BBC biochemical, USA). Sections of paraffin-embedded tissues were stained with hematoxylin (Merck, USA) and eosin (Merck, USA) and examined under a ScanScope XT virtual digital slide scanner (Aperio, USA) (magnification, ×200). The magnified slide was visualized using Imagescope software version 11.2.0.782 (Aperio, USA). Sections of paraffin-embedded organs containing metastatic deposits were examined by immunofluorescence using a Zeiss AxioImager M1 Fluorescence microscope (Carl Zeiss, Germany).

For in vitro staining of Spike protein expression 293T cells were cultured on 4-well glass slides (Lab-Tek) and transfected with 3 µg per well of pDNA using TurboFectin8.0 (OriGene) transfection reagent following the manufacturer’s protocol. Cells were fixed 48 h after transfection with 10% Neutral-buffered Formalin (BBC Biochemical, Washington State) for 10 min at RT and then washed with PBS. Before staining, chamber slides were blocked with 0.3% (v/v) Triton-X (Sigma), 2% (v/v) donkey serum in PBS for 1 h at RT. Cells were stained with a rabbit anti-SARS-CoV spike protein polyclonal antibody (Novus Biologicals) diluted in 1% (w/v) BSA (Sigma), 2% (v/v) donkey serum, 0.3% (v/v) Triton-X (Sigma) and 0.025% (v/v) 1 g ml⁻¹ Sodium Azide (Sigma) in PBS for 2 h at RT. Slides were washed three times for 5 min in PBS and then stained with donkey anti-rabbit IgG AF488 (lifetechnologies, A21206) for 1 h at RT. Slides were washed again and mounted and covered with DAPI-Fluoromount (SouthernBiotech).

Tissue preparation was carried out as described [10 (link)]. Briefly, animals were deeply anesthetized and perfused with heparinized PBS. The eyes and optic nerves were extracted separately by cutting the optic nerve approximately 0.5 mm from the posterior pole of the eyeball. Low-temperature cautery was used to mark the 12-o’clock position on each cornea and both eye and optic nerve specimens were fixed in 10% neutral buffered formalin (BBC Biochemical, Everett, WA) at 4°C for 24 hours and then transferred into 80% ethanol for long-term storage. Samples were processed for paraffin embedding using a standard protocol and embedded in paraffin in such a way as to ensure a cut through the vertical meridian of the eyeball and a longitudinal cut through the optic nerve.
Paraffin blocks were sectioned at a thickness of 4 microns and mounted on positively charged glass slides (Erie Scientific, Portsmouth, NH).
Hematoxylin and eosin (H&E) staining was applied to the optic nerves and eye cross sections. In addition, Luxol fast blue (LFB) staining with cresyl violet (CV) as a counterstain was applied to the optic nerve cross sections.

The tumors obtained from the vehicle and treatment groups of mice were fixed with neutral buffered formalin (10%, BBC Biochemical, Mount Vernon, WA, USA). They were processed and made into paraffin blocks. Then, IHC was performed as described earlier [54 (link)].

The tissues were fixed in 10% neutral-buffered formalin (BBC Biochemical, Mount Vernon, WA, USA) and embedded within paraffin. The paraffin-embedded samples were sliced into 5 μm sections and histological observation was performed following hematoxylin and eosin (H&E) staining. For immunohistochemistry, tissue sections were stained with an MMP-1 antibody (ab52631, Abcam, Cambridge, MA, USA) at 4 °C overnight. The expression level of MMP-1 in the dermis was quantified by using Image-Pro Plus 7 software (Media Cybernetics, Inc. Rockville, MD, USA).