Neutral buffered formalin solution

Fifteen Holstein-Friesian cows, of mixed parity, within the same university dairy herd were sampled 7 and 21 days postpartum (DPP) in the morning after milking. A clinical examination and sample collection were conducted by a veterinarian and involved recording the rectal temperature, body condition score, heart rate and vaginal discharge. At each time-point, an endometrial biopsy was taken from the same post-gravid horn as previously described [67 (link)]. Uterine culture swabs were placed (in duplicate per cow) in 1 ml of Tris-EDTA buffer (Sigma Aldrich® Vale Road, Arklow, Wicklow, Ireland). Immediately after collection, the biopsy was dissected in two - one half was snap frozen in liquid nitrogen for transcriptomic analysis, and the other half was fixed in 10 % neutral-buffered formalin solution (Sigma Aldrich® Vale Road, Arklow, Wicklow, Ireland) for histopathological assessment. Whole blood was collected in spray-coated K₂EDTA Vacutainer®, for haematology and plasma protein analysis, followed by 4 ml of blood in one sodium fluoride/Na₂ EDTA Vacutainer®, for blood metabolite analysis at pre- and postpartum time points. All experimental procedures were carried out under license from the Irish Department of Health and Children in accordance with the European Community Directive 86-609-EC and were approved by the Animal Research Ethics Committee, University College Dublin.

The specimen including the implant was prepared after the experimental animals were sacrificed. The specimen was fixed for 2 weeks in a neutral buffered formalin solution (Sigma-Aldrich, St. Louis, MO, USA) and then dehydrated by increasing the ethanol concentration from 70 to 100%. The recovered formalin-fixed alcohol-preserved specimens were decalcified in 10% formic acid/formalin solution for 14 days, dehydrated, paraffin-embedded, microsected parallel to the bone axes, and stained with hematoxylin and eosin (H&E) and Masson's trichrome. Histological observation of the grafted bone granule resorption, degree of regenerated bone replacement, and the inflammatory response were recorded using Olympus BX40 microscopy (Olympus, Tokyo, Japan).

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), and bilirubin were measured in serum with the UniCel® DxC 800 Synchron® Clinical System (Beckman Coulter GmbH).
One-half of the right liver lobe was fixed in 10% neutral buffered formalin solution (Sigma Aldrich, Germany) and embedded in paraffin. Five μm thin sections were stained with either hematoxylin/eosin (HE) or Sirius red (SR).

Following transcardial perfusion, the brains were collected and post-fixed in 10% neutral-buffered formalin solution (Sigma Chemical Co., St. Louis, MO, USA) for 48 hours. The two hemispheres of each brain were separated and embedded in paraffin (Sigma Chemical Co., St. Louis, MO, USA). The brains were cut sagittally at a 5 µm thickness and mounted on silane-coated slides (Superfrost Plus Micro Slide, VWR, Arlington Heights, IL, USA). Histologic monitoring of myelin was performed using Luxol fast blue staining as previously described^{45 (link)}. Images of the lesion area were acquired with a camera-equipped light microscope (Axio Observer A1. Zeiss Microscope) using AxioVision software. Three brain sections from the center of the demyelination lesion of 4–7 different rats per group were acquired at 10x magnification and used for the image analysis. The demyelinated area was delineated and measured using ImageJ software^{48 (link)}. The obtained values of lesion size were used as an index of de/remyelination as previously described^{49 (link)}.

Immunostaining of murine skin and gut specimens was carried out on sections from paraffin-embedded tissues fixed in 10% neutral-buffered formalin solution (Sigma-Aldrich, St. Louis, MO) using streptavidin-biotinylated HRP detection (Beckeman Coulter, Brea, CA) as previously described with slight modification [20] (link). For antigen retrieval, sections (3.5 µm thickness) on silane-coated slides were heated in a microwave oven for 45 minutes at 98°C in immunosaver (1∶200 dilution, Nisshin EM Corp. Tokyo, Japan). After blocking nonspecific binding with normal rabbit serum (1∶75 dilution; Dako Inc. Via Real (Carpinteria, CA), sections were incubated with an anti F4/80 mAb (CI:A3-1, Novus, Littleton, CO) (1∶100) or an isotype-matched mAb for 15 minutes using intermittent microwave irradiation [21] (link), [22] (link). Sections were then incubated with biotin-labeled rabbit anti-mouse IgG pAb (1∶300 dilution; Dako Inc.) and 3,3′-diaminobenzidine (DAB; Vector Laboratories Inc. Burlingame, CA) was used as chromogen. Finally they were counterstained with hematoxylin.