Nandini Narayan

• Flash freezing freshly harvested mouse samples for cryo- sectioning
• Cryo- sectioning a variety of fresh- frozen neuromuscular specimens including whole brain slices, diaphragm, tibialis anterior, gastrocnemius and cardiac muscle
• Optimizing and manually performing single-plex and multiplex immunofluorescence staining on fresh frozen specimens. Multiplex assays include the use of the Streptavidin- Biotin complex whenever applicable
• Manually running the RNAscope (ISH) assays including the multiplex fluorescent RNAscope, BaseScope, miRNAscope and smRNA- RNAscope assays on FFPE and fresh- frozen specimens.
• Brightfield and fluorescent scanning of slides on the Zeiss AxioScan Z1 digital scanner
• Performing quantitative image analysis on scanned slides using the HALO platform
• Presenting data from histological end point assays in interdepartmental meetings
• Co-ordinates with CROs for running routine and special stains on rodent samples
• Training new personnel on sample collection, cryo-sectioning and staining techniques

• Cutting a variety of FFPE and frozen specimens for staining and digital scanning.
• Manually staining specimens for Hemotoxylin and Eosin (H&E) as well as special stains such as Masson’s Trichrome, Oil Red O as requested by the client.
• Performing single-plex Immunohistochemistry (IHC) on the Leica Bond as well as optimizing IHC protocols for new biomarkers on the Leica Bond.
• Performing single-plex and multiplex immunofluorescent (IF) assays manually as well on the Leica Bond. Multiplex assays include the use sequential staining using the Tyramide Signal Amplification (TSA) assay whenever applicable.
• Performing routine maintenance on the automated stainers, tissue processor, embedding station, microtomes and cryostat.
• Brightfield and fluorescent scanning of client slides on the 3D Histech Pannoramic Scan II platform, reviewing scanned images and rescanning and trouble-shooting as needed.

• Tissue Micro-dissection or scraping the tumor and benign region of interest (ROI) from FFPE tissue sections for further
  molecular testing that identifies the oncogene in patients.
• Scanning and reviewing fluorescently labeled breast cancer patient slides for four biomarkers: estrogen receptor (ER),
  progesterone receptor (PR), human epidermal growth factor receptor 2 (Her2) and proliferative biomarker Ki67 using
  the Aperio Scanscope and Perkin Elmer Vectra digital slide scanning platforms.
• Staining all specimens for hemotoxylin and eosin (H&E) in the Sakura Tissue-Tek® Prisma® Automated Slide Stainer for pathological review to determine tumor content before proceeding with further testing.
• Immunohistochemical staining breast tissue for ER,PR HER2 and Ki67 using the Ventana Discovery Ultra platform.
• Updating Laboratory Information System (LIS) for all incoming specimens on a daily basis, writing quality control
  documents for training personnel, and modifying existing documents on a quarterly basis.
• Accessioning FFPE blocks, clinical trial specimens, assigning the appropriate tests to the incoming specimens and sending them other departments, if necessary.
• Etching of slides using diamond pen for FISH analyses and for other cytogenetic markers
• Writing Operational qualification, Instrument qualification and Performance qualification protocols for lab equipment

• Processing of murine pancreas, spleen and kidney samples for paraffin embedment and sectioning for histochemical analysis.
• Chromogenic and fluorescent histochemical/immunohisotchemical staining of specimens for different biomarkers such as Insulin/Ki67, Glucagon/Ki67, alpha and beta islet cells in pancreas and kidneys
• Slide Scanning and image analyses using the Nanozoomer Digital Pathology System

SCIENTIFIC PAPERS

IL-2 Immunotherapy Reveals Potential for Innate Beta Cell Regeneration in the Non-Obese Diabetic Mouse Model of

Autoimmune Diabetes

Type-1 diabetes (T1D) is an autoimmune disease targeting insulin-producing beta cells, resulting in dependence on exogenous insulin. To date, significant efforts have been invested to develop immune-modulatory therapies for T1D treatment. Previously, IL-2 immunotherapy was demonstrated to prevent and reverse T1D at onset in the non-obese diabetic (NOD) mouse model, revealing potential as a therapy in early disease stage in humans. In the NOD model, IL-2 deficiency contributes to a loss of regulatory T cell function. This deficiency can be augmented with IL-2 or antibody bound to IL-2 (Ab/IL-2) therapy, resulting in regulatory T cell expansion and potentiation. However, an understanding of the mechanism by which reconstituted regulatory T cell function allows for reversal of diabetes after onset is not clearly understood. Here, we describe that Ab/IL-2 immunotherapy treatment, given at the time of diabetes onset in NOD mice, not only correlated with reversal of diabetes and expansion of Treg cells, but also demonstrated the ability to significantly increase beta cell proliferation. Proliferation appeared specific to Ab/IL-2 immunotherapy, as anti-CD3 therapy did not have a similar effect. Furthermore, to assess the effect of Ab/IL-2 immunotherapy well after the development of diabetes, we tested the effect of delaying treatment for 4 weeks after diabetes onset, when beta cells were virtually absent. At this late stage after diabetes onset, Ab/IL-2 treatment was not sufficient to reverse hyperglycemia. However, it did promote survival in the absence of exogenous insulin. Proliferation of beta cells could not account for this improvement as few beta cells remained. Rather, abnormal insulin and glucagon dual-expressing cells were the only insulin-expressing cells observed in islets from mice with established disease. Thus, these data suggest that in diabetic NOD mice, beta cells have an innate capacity for regeneration both early and late in disease, which is revealed through IL-2 immunotherapy.

• Analyzing patient data and samples from the Moffit Cancer Center in Tampa, Florida to understand the role of Akt kinases in tumor development
• Scoring breast, colon, prostate, and lung cancer patient samples from for cytoplasmic and/or membrane signal intensities using biomarkers such as IGF1R and pS6
• Sectioning and paraffin embedment of murine and patient tissue samples for Immunohistochemical analyses
• Employing various benchtop and automated IHC staining techniques for membrane and cytoplasmic analyses of different tissues to study antibody affinity for different biomarkers
• Maintaining a detailed electronic lab notebook, submitting biweekly progress reports and presenting project data to senior management