Dialysis tubing

Glucose, acetic acid, sodium hydroxide (NaOH), citric acid, ammonia, hydrochloric acid (HCl), potassium dichromate and ascorbic acid were delivered by POCH, Gliwice, Poland. Glucosamine, chitosan, cellulose, XTT assay and PBS (phosphate buffer solution) was delivered by Sigma-Aldrich, Poznań, Poland. Dulbecco’s modified Eagle medium (DMEM), streptomycin/penicillin and fetal bovine serum was delivered by Polgen, Łódź, Poland. Dialysis tubing (molecular weight cut off (MWCO) 500–1000 Da) was purchased from VWR, Gdańsk, Poland.

Fluorescein was covalently coupled to KLH and to BSA to provide a T-dependent antigen for immunization and an antigen for use in ELISA assays, respectively. To label KLH (Sigma-Aldrich, St. Louis, MO, USA) with FITC (Sigma-Aldrich, St. Louis, MO, USA), 1.5mL of 10mg/mL KLH was first dialyzed against 2L of 100mM NaCO₃ buffer at pH 9.3 in dialysis tubing (MWCO: 3,500; VWR Scientific, Rancho Dominguez, CA, USA) at 4°C for 24 hrs with one change in dialysis solution during this process. FITC and KLH were then combined at a 20μg:1mg ratio, and incubated for 1.5 h at room temperature. Next, the FITC-KLH conjugate was dialyzed in 500mL saline solution overnight at 4°C. The buffer was changed, and the sample was dialyzed for 2 hrs at room temperature. The absorbance of purified FITC-KLH was read at 280nm and 495nm, and the labeling ratio (FITC:Protein) was determined based on the ratio of those absorbances. Two batches, with labeling ratios 2.4 and 5.3, were made and pooled. BSA (Roche, Indianapolis, IN, USA) was labeled using the same protocol, with a molecular labeling ratio of 1:4. Both labeled proteins, FITC-KLH and FITC-BSA, were stored at -80°C until use. Labeled proteins were stored so that each aliquot was thawed only once prior to use.

Imidazole, Trizma base and acid, DNase, SIGMAFAST protease tablets, NaCl, Ni²⁺-resin, anti-PKP1 antibody, DAPI (4′,6-diamidino-2-phenylindole), paraformaldehyde (PFA), and ultra-pure dioxane were obtained from Sigma (Madrid, Spain). Ampicillin, kanamycin, and isopropyl-β-D-1-thiogalactopyranoside were obtained from Apollo Scientific (Stockport, UK). Dialysis tubing with a molecular weight cut-off of 3500 Da, Triton X-100, TCEP (tris(2-carboxyethyl)phosphine) and the SDS protein marker (PAGEmark Tricolor) were obtained from VWR (Barcelona, Spain). Amicon centrifugal devices with a molecular weight cut-off of 30 kDa were obtained from Millipore (Barcelona, Spain). The rest of the materials were of analytical grade. Water was deionized and purified on a Millipore system.