Comprehensive Evaluation of PAXgene Fixation on Oral Cancer Tissues Using Routine Histology, Immunohistochemistry, and FTIR Microspectroscopy

Abstract

The choice of tissue fixation is critical for preserving the morphology and biochemical information of tissues. Fragile oral tissues with lower tensile strength are challenging to process for histological applications as they are prone to processing damage, such as tissue tear, wrinkling, and tissue fall-off from slides. This leads to loss of morphological information and unnecessary delay in experimentation. In this study, we have characterized the new PAXgene tissue fixation system on oral buccal mucosal tissue of cancerous and normal pathology for routine histological and immunohistochemical applications. We aimed to minimize the processing damage of tissues and improve the quality of histological experiments. We also examined the preservation of biomolecules by PAXgene fixation using FTIR microspectroscopy. Our results demonstrate that the PAXgene-fixed tissues showed significantly less tissue fall-off from slides. Hematoxylin and Eosin staining showed comparable morphology between formalin-fixed and PAXgene-fixed tissues. Good quality and slightly superior immunostaining for cancer-associated proteins p53 and CK5/6 were observed in PAXgene-fixed tissues without antigen retrieval than formalin-fixed tissues. Further, FTIR measurements revealed superior preservation of glycogen, fatty acids, and amide III protein secondary structures in PAXgene-fixed tissues. Overall, we present the first comprehensive evaluation of the PAXgene tissue fixation system in oral tissues. This study concludes that the PAXgene tissue fixation system can be applied to oral tissues to perform diagnostic molecular pathology experiments without compromising the quality of the morphology or biochemistry of biomolecules.

FTIR microspectroscopy and multivariate analysis facilitate identification of dynamic changes in epithelial‐to‐mesenchymal transition induced by TGF‐β in prostate cancer cells

“…Collected spectra were smoothened using Savitzky–Golay filter of polynomial order 2 with 10 smoothening points. Spectral images from the specific bio‐fingerprint region were used to recognize discriminative molecular vibrations unique to various treatment conditions 15 . Operations such as smoothening, detecting and labeling IR peaks, and identifying their absorbance values were carried out with SpectraGryph v.1.2.13 software, while OriginPro 2019b was used for principal component (PCA) and hierarchical cluster analyses (HCA).…”

“…Spectral images from the specific bio-fingerprint region were used to recognize discriminative molecular vibrations unique to various treatment conditions. 15 Operations such as smoothening, detecting and labeling IR peaks, and identifying their absorbance values were carried out with SpectraGryph v.1.2.13 software, while OriginPro 2019b was used for principal component (PCA) and hierarchical cluster analyses (HCA). Dendrograms were produced using Ward's clustering method with a correlation distance matrix to identify the similarities between the various phenotypic (cell morphology, migration, invasion, and colony formation) and metabolic features (saturation, unsaturation, levels of Nicotinamide adenine [NADH] and FADH2).…”

Local metabolism in preclinical disease models studied with mass spectrometry imaging

“…In parallel, new preservation compounds are being developed that bridge the necessities in biomedical research with established clinical workflows. Such an example is PaxGene, which has already been used in MSI studies for the investigation of proteins, peptides and metabolites [486] and shows good compatibility with the requirements from clinical practice [487]. The further investigation of such alternatives for tissue preservation also carries huge potential for the broadness and depth of molecular information that can be extracted from preserved tissues using MSI.…”