Sirius red fast green collagen staining kit

mPSC (BALB/c) and hPSCs (40,000) were grown in a 24-well plate for 24 h prior to 24 h stimulation with agonist or inhibitor and 5 μM Aphidicolin (Sigma) in 0% FBS media. Collagen was detected with the Sirius Red/Fast Green Collagen Staining kit (Chondrex, Redmond, WA, USA). Absorbance was measured in a FLUOstar Optima microplate reader and the extracellular collagen levels were calculated according to the manufacturer’s instructions.

Collagen content was quantified using a Sirius Red/Fast Green Collagen Staining Kit (Chondrex, Redmond, WA) according to the manufacturer's instructions. First, two paraffin sections of spheroids (on one slide) were deparaffinized and transferred to an individual petri dish. A 0.3 ml aliquot of Sirius Red/Fast Green dye solution was added to each petri dish to immerse the sections, and then they were incubated for 30 min at room temperature. After incubation, the dye solution was carefully removed, and the slides were rinsed with tap water until no color was observed in the water. Each slide was subsequently transferred to a new petri dish and covered with 1 ml of dye extraction buffer from the kit. The dye was extracted from the stained samples with gentle shaking and pipetting. Then, 200 µl of eluted dye solution was transferred to a 96-well plate for optical density (OD) reading at 540 nm and 605 nm using a microplate reader. The collagen content in each sample was normalized to the total protein content.

To quantify collagen content in melanoma tissues, a Sirius Red/Fast Green Collagen Staining Kit (Chondrex, Redmond, WA) was used according to the manufacturer’s instructions. Briefly, after the slide was incubated with the dye solution at room temperature for 30 minutes, the dye solution was removed, and the slide was rinsed with distilled water until the water became colorless. One milliliter of dye extraction buffer was added to each slide to elute the dye from dyed tissues. A 200 μl dye extraction solution from each slide was collected for absorbance measurements at 540 nm and 605 nm using a microplate reader. The collagen content in each sample was normalized to the total protein content.

Staining was performed using a Sirius Red/Fast Green Collagen Staining Kit (9046, Chondrex) following the manufacturer's recommendations.

The mouse lungs were inflated with a neutral buffered formalin solution overnight and embedded in paraffin before sectioning into 5 μm-thick slices. The sections were stained with hematoxylin–eosin for structure observation, or used for detection of collagen deposition by Sirius Red/Fast Green Collagen Staining Kit (Chondrex, MA).

The total protein content, and the fibrillar collagen content of the deposited matrices, was measured with the Sirius red/fast green collagen staining kit (Chondrex Inc., Redmond, USA) following the manufacturer’s specification. The optical density values (OD) of eluted dye were measured at 540 nm and 605 nm with the Synergy^TMHT multi-mode microplate reader (Biotek Instruments Inc.). Calculations were made with the formulas: total protein = {[OD₅₄₀ −  (OD₆₀₅ × 0.291)]/0.0378} + OD₆₀₅/0.00204; collagen = [OD₅₄₀ − (OD₆₀₅ × 0.291)]/0.0378.

Mouse livers were fixed in 10% formalin overnight. Paraffin-embedded sections were stained with hematoxylin and eosin (H&E) or assessed for collagen content by following the manufacturer’s protocol [39 ] in use of a Sirius Red/Fast Green collagen staining kit (#9046, Chondrex, Redmond, WA, USA). In short, deparaffinized tissue sections were incubated with the reaction buffer containing both Sirius Red and Fast Green dyes for 30 min. After incubation, the sections were rinsed repeatedly with water until the water ran clear. The Sirius Red dye binds to collagen and the Fast Green dye binds to non-collagenous proteins. After the bright field images were captured, both dyes were eluted from the sections using extraction buffer and the absorption of eluted dye at 540 nm (Sirius Red) and 605 nm (Fast Green) was measured. The ratio of optical density (OD) values was used as a semiquantitative estimation of collagen and non-collagenous protein in each section. Blood chemistry analysis was performed using the DRI-CHEM 3500s (Fuji Film, Tokyo, Japan) and the Fuji Dry-Chem Slide (TCHOP-P III).