Serum Albumin

Two flowers per day from anthesis, two and three days after pollination were fixed in 4% formaldehyde freshly prepared from paraformaldehyde in 1x phosphate saline buffer (PBS) pH7.3, left overnight at 4ºC, and conserved then at 0.1% formaldehyde solution [83 (link)]. Then the pistils were dehydrated in an acetone series (30%, 50%, 70%, 90%, 100%), and embedded in Technovit 8100 (Kulzer and Co, Germany) for two days. The resin was polymerized at 4ºC, and sectioned at 4 μm thickness. Sections were placed in a drop of water on a slide covered with 2% (3-Aminopropyl) triethoxysilane - APTEX (Sigma-Aldrich), and dried at room temperature. Callose was identified with the anticallose antibody (AntiCal) that recognises linear β-(1,3)-glucan segments (anti-β-(1,3)-glucan; immunoglobulin G1), Biosupplies, Australia [49 (link)]. As a secondary antibody, Alexa 488 fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG was used (F-1763; Sigma). Additionally, a monoclonal antibody (mAbs) JIM13 [84 (link)] against AGPs glycosyl epitopes, and one mAb JIM11 [85 (link)] against extensin epitopes were obtained from Carbosource Services (University of Georgia, USA). Secondary antibodies were anti-rat IgG conjugated with the same Alexa 488 used above. Sections were incubated for 5 min in PBS pH7.3 followed by 5% bovine serum albumin (BSA) in PBS for 5 min. Then, sections were incubated at room temperature for 1h with AntiCal primary mAb, JIM13, and JIM11. After that, three washes in PBS of 5 minutes each preceded the incubation for 45 min in the dark with a 1/25 diluted secondary fluorescein isothiocyanate (FITC) conjugated with the antibody in 1% BSA in PBS, followed by three washes in PBS [83 (link)]. Sections were counterstained with calcofluor white for cellulose [86 (link)], mounted in PBS or Mowiol, and examined under a LEICA DM2500 epifluorescence microscope connected to a LEICA DFC320 camera. Filters were 355/455 nm for calcofluor white and 470/525 nm for the Alexa 488 fluorescein label of the antibodies (White Level?=?255; Black Level = 0; ϒ?=?1). Exposur (Exp) times were adapted to the best compromise in overlapping photographs for each antibody: AntiCal, Exp.?=?15.30ms (Calcofluor Exp. = 1.20ms); JIM13 Exp.?=?2.52ms (Calcofluor?=?0.41ms); JIM11, Exp. = 31.59 ms (Calcofluor Exp. = 1.40ms). Brightness and contrasts were adjusted to obtain the sharpest images with the Leica Application Suite software.

The eyeballs of rats were fixed with 4% paraformaldehyde for 2 h, then embedded with optimal cutting temperature compound (OCT) at –80 °C and cut into 8-μm-thick sections for their immunofluorescence staining. Cryosections were washed twice with PBS, permeabilized with 0.3% Triton X-100 in PBS, and blocked in 0.1% Triton X-100 and 2% bovine serum albumin (BSA) in PBS. The blocked corneal tissue was then incubated overnight at 4 °C with primary antibodies: these were p38, phospho-p38 MAPK (Thr180/Tyr182), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), JNK, and p-JNK (Thr183/Tyr185) (Cell Signaling Technology; Danvers, MA, USA). After the corneal cells were incubated with secondary antibodies, their nuclei were counterstained with DAPI. All sections were detected using CLSM (LSM 800, Zeiss, Germany).

Detailed baseline and clinicopathological information, including sex, age, tumor location, tumor size, pathological type, differentiation, lymph node metastasis, and TNM stage of the patients with pancreatic diseases and HC, were obtained from the medical records of the inpatients or outpatients. The preoperative hematological parameters and liver function tests included neutrophils (× 10⁹/L), lymphocytes (× 10⁹/L), monocytes (× 10⁹/L), platelets (× 10⁹/L), plasma fibrinogens (g/L), serum albumins (g/L), prealbumin (mg/L), and CA199 (U/L) within seven days before surgery (average 2—7 days) were gathered from the medical records. TNM staging was performed using the 8th edition of the AJCC Cancer Staging Manual for Pancreatic Cancer.

FAR, FPR, NLR, PLR, MLR, and FLR were defined as the plasma fibrinogen value divided by the serum albumin value, plasma fibrinogen value divided by the serum prealbumin value, neutrophil count divided by the lymphocyte count, platelet count divided by the lymphocyte count, monocyte count divided by the lymphocyte count, and plasma fibrinogen value divided by the lymphocyte count, respectively. PNI was defined as serum albumin value + 5 × lymphocyte count.