Naphtol as mx phosphate

Acetone-fixed, 5-μm cryosections of spleens and livers were stained for immunohistochemistry as described previously (27 (link)). Briefly, sections were rehydrated in TBS (pH 7.6) and incubated with primary Abs at room temperature for 45 min. The primary Abs used their sources and concentrations can be found in Supplemental Table I. HRP-conjugated or biotin-conjugated secondary Abs and ABComplex Alkaline Phosphatase (Dako) were used to detect labeled cells. HRP activity was detected with SIGMAFAST 3-3′Diaminobenzidine Tablets, whereas alkaline phosphatase activity was detected using Naphtol AS-MX Phosphate and fast blue salt with levamisole (all from Sigma-Aldrich). Staining for immunofluorescence was performed as described (28 (link)). Briefly, sections were rehydrated in PBS (pH 7.4) and incubated with primary Abs in the dark at room temperature for 40 min. Secondary Abs were used in all cases and were added for 30 min at room temperature in the dark. The details of primary and secondary Abs used are in Supplemental Table I. After staining, slides were mounted in Prolong Diamond (Invitrogen).

The 6-well plates were removed from the incubator and the cells carefully washed with 500 µL PBS. The cells were fixed with 400 µL 10% glutaraldehyde solution. The glutaraldehyde solution was removed, and the cells were washed with 400 µL PBS at least three times. The cells were coated with 400 µL freshly prepared TRAP solution (0.3 mg Fast Red Violet LB stain (F-3381, Sigma-Aldrich, St. Louis, MO, USA) per mL TRAP Buffer (50 mL 0.1 M Acetate Buffer, 10 mL 0.3 M Sodium Tartrate, 1 mL 10 mg/mL Naphtol AS-MX Phosphate (N-5000, Sigma-Aldrich, St. Louis, MO, USA), 100 µL Triton X-100, 38.9 mL H₂O_dd) and incubated at 37 °C for 15 min. Again, the cells were washed twice with 400 µL PBS. The TRAP⁺-cells were quantified in 17 visual fields under the microscope (Olympus IX50).

The cell-seeded cryogels were fixed for 2 min with 4% PFA. Afterwards, the scaffolds were washed with PBS and could be stored at 4 °C. Before staining, the scaffold was rinsed with 0.1 M Tris HCl (pH = 9.2, Trizma base minimum (Sigma Aldrich, Steinheim, Germany) in deionized water). Then, the following substances were also dissolved in 10 mL Tris HCl in order to prepare the staining solution: 2 mg of naphtol AS-MX phosphate (Sigma Aldrich, Steinheim, Germany) and 10 mg of Fast Red TR (Sigma Aldrich, Steinheim, Germany). An appropriate volume was transferred into every well and the samples were incubated for 20 min in a shaking device. At the end, the red precipitate was analyzed on a brightfield microscope Olympus IX73 (Olympus Life Science).

On day 43, mice were sacrificed and knees joints were removed and fixed in 4% formalin. Paws were decalcified in 10% EDTA for 30 days and, then, were dehydrated and embedded in paraffin. Samples sectioned (7 mm) were stained with hematoxylin and eosin (H&E) or tartrate-resistant acid phosphate (TRAP) using naphtol-AS-MX phosphate (Sigma^®, St. Louis, MO, USA) and fat red violet LB salt (Sigma^®, St. Louis, MO, USA), to perform histological analysis.

Tartrate-resistant Acid Phosphatase (TRAP) staining was performed (28 (link)). Caudal fins of adult WT (n = 7), tmem38b^-/- (n = 9) and tmem38b^{Δ120-7/Δ120-7} (n = 10) zebrafish were fixed in PFA 4% in PBS o/n at 4°C, washed in PBS containing 0.1% v/v Tween-20 and permeabilized in PBS containing 0.3% v/v Triton X-100 for 30 min. Fins were then equilibrated in TRAP buffer (0.1 M sodium acetate, 0.1 M acetic acid, 50 mM sodium tartrate) and colour reaction was performed in TRAP buffer containing 0.1 mg/ml Naphtol AS-MX phosphate (Sigma Aldrich) and 0.3 mg/ml Fast Red Violet LB (Sigma Aldrich). Fins were then bleached in 10% H₂O₂, 1% KOH o/n at RT to remove pigmentation and then stored in 70% glycerol at 4°C. Images were acquired using a Leica M165 FC microscope connected to a Leica DFC425 C digital camera. The number of TRAP+ cells in the regenerate was counted using the Cell Counter tool on the ImageJ software according to literature (29 (link)).