Silver nitrate solution

The rapid Golgi impregnation used two solutions as follows:
Solution A. 3% Potassium dichromate solution (Sigma-Aldrich, no. P5271, USA) stirred into warm deionised water.
Solution B. 2% Silver nitrate solution (Sigma-Aldrich, no. 20,913-9, Germany) stirred into deionised water.
These solutions are never mixed with each another.
The tissues were placed 3% potassium dichromate Solution for 7 days in the dark, changing solutions daily using glass pipettes, and then transferred into 2% Silver nitrate solution for 3 days in the dark at room temperature.

Following the second chemical treatment, cells were harvested as previously outlined, for tissues treated topically with 0, 1000 and 1900 µg/ml MMS. Following centrifugation at 100 × g, supernatant was removed and total RNA extracted from the pellet using the RNeasy Kit (Qiagen, Crawley, UK) and RNase-free DNase Set (Qiagen). Eluted RNA solution was quantified using the NanoDrop (ND 1000) Spectrophotometer, software version 3.1.2. Synthesis of complementary DNA (cDNA) was performed via reverse transcription of 1 µg RNA using the Quantitect Reverse Transcription Kit (Qiagen) and BioRad T100 Thermal Cycler using conditions specified by the manufacturer. The polymerase chain reaction (PCR) was performed for each cDNA sample, using the GoTaq® Flexi DNA Polymerase kit (Promega, Southampton, UK). N-methylpurine-DNA glycosylase (MPG) primers (Sigma-Aldrich): Forward primer: GGTCCGAGTCCCACGAAGCC. Reverse primer: CTGCATGACCTGGGCCCCG. β–actin (housekeeping gene): Forward primer: GATGGCCACGGCTGCTTC. Reverse primer: TGCCTCAGGGCAGCGGAA. Polyacrylamide gels (5.4%) were cast and DNA polyacrylamide gel electrophoresis (PAGE) performed at 170V for 30min. DNA was stained by washing the gel for 7min in 1g/l silver nitrate solution (Sigma-Aldrich), followed by a 3min wash in sodium hydroxide/formaldehyde solution (Sigma-Aldrich) until the bands were visible, followed by submersion in ddH₂O.

The ion-exchange resins’ evaluation by analysis of their residual water hardness was supplemented by control of the brine parameters after regeneration. Reagents for the method—consisting of silver nitrate solution as titrant and potassium chromate as indicator solution—were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water was obtained from an in-house industrial deionization installation (Sanhua-Aweco, Tychy, Poland).
The method was used in accordance with Standard Methods for the Examination of Water and Wastewater [24 ].

Von Kossa staining, used to identify osteoblast
cells, was conducted on days 1, 14 and 21. Approximately,
10% (v/v) formalin (R&M Chemicals, UK)
in phosphate buffer saline (PBS, Sigma, USA) was
added into each well and incubated for 30 minutes.
Then, cultured cells were washed with deionized water
3 times, followed by cells incubation with 100%
(v/v) silver nitrate solution (Sigma, USA) for 30 minutes
in dark room. After this cells were washed thrice
with deionized water, followed by the addition of
10% (w/v) sodium carbonate (Fisher Scientific, UK)
solution in 25% (v/v) formalin, it was incubated for 5
minutes at room temperature. Thereafter, the cultured
cells were washed with deionized water three times
before the addition of 5% (w/v) sodium thiosulphate
(Sigma, USA) solution. After 2 minutes, cultured
cells were washed again with deionized water three
times. Cells which were able to differentiate to mature
osteoblast cells were stained by von Kossa staining.
Characteristically in this technique, calcium materials
are identifiable by black or brown color deposits,
so osteoblast cells were visualized as brown or dark brown
color.

Poly(allylamine hydrochloride) (Mw 56,000), poly(acrylic acid, sodium salt) 35 wt.% solution in water (PAA) (Mw 15,000), silver nitrate solution (> 99% titration, 0.1 N AgNO₃), and dimethylamine borane complex (DMAB) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without any further purification. Aqueous solutions of 0.01 M of both PAH and PAA were prepared using ultrapure deionized water (18.2 MΩ) and adjusted to pH values 7.0 and 9.0 by the addition of a few drops of HCl or NaOH 1 M.