This document discusses optimizing staining techniques to visualize different cell types in vitro. It examines Giemsa May-Grundwald staining, Rhodamine phalloidin to visualize the cytoskeleton, and immunohistochemistry using antibodies against specific proteins. The aim was to identify the best staining and imaging conditions for visualizing the cytoskeleton of Neuro-2A, BEAS-2B, and A549 cells grown in vitro. Results found that immunofluorescence staining provided better cytoskeleton imaging than colorimetric methods, with different cell types staining equally well. Phalloidin also appeared to offer better actin staining than Neuro-D1. Future work proposed investigating the impact of graphene oxide on cells using these techniques.

1. NeurN
CONCLUSIONS
INTRODUCTION
RESULTS
REFERENCES
The suitability of various techniques
for the in vitro imaging of different cell types
Thelwall, I.
Nanomedicine Lab, School of Health Sciences & National Graphene Institute, The University of Manchester, UK
iona.thelwall@manchester.ac.uk
Histology can be defined as the microscopic study of cells and tissues via the
staining and sectioning of samples. There have been significant changes to
histological staining techniques (Alturkistani et al. 2016). Ranging from the use of
readily available chemicals, such as Giemsa, to the introduction of immunological
techniques.
Giemsa May-Grundwald staining has been utilised since the 1890s and is still
fundamental in clinical practice (Musumeci et al. 2014). Rhodamine phalloidin is a
phallotoxin used to visualise the cytoskeleton which forms tight complexes
with F-actin (Wulf et al. 1979). Finally, immunohistochemistry is an advanced
histological technique which emerged in the 1980s and utilises antibodies against
specific proteins found in the cell (Musumeci et al. 2014).
AIM & OBJECTIVES
Specific Objectives:
v Optimise different staining to visualise the cytoskeleton
v Investigate the effectiveness of different staining methods for cell
imaging
v Determine the impact of the different cell types on the images obtained
vGiemsa May-Grundwald staining of different cell types
v The Neuro-D1 staining did not work, however the merge includes NeurN staining.
v The negative control shows no auto-fluorescence suggesting that all of the
proteins which have successfully stained are present.
MAP2
v Antibody staining of Neuro2A cells
v Antibody and Phalloidin staining of BEAS2B cells
v The negative control appears to show no auto-fluorescence.
v The LAMP1 staining is in the correct area, but is fainter than expected.
Overarching Aim
The main aim of this work was to identify the best staining and imaging
conditions to visualise the cytoskeleton of cells grown in vitro
v Immunofluorescence staining provides better imaging of the
cytoskeleton than colorimetric method. Different cell types stained
equally well.
v Phalloidin appeared to offer better actin staining than Neuro-D1.
v Lysosomal staining was challenging even after exposure to graphene
oxide. Leading us to believe that the LAMP1 antibody may not
bespecific enough.
FUTURE WORK
v In the future it would be interesting to use these techniques to
investigate the impact of graphene oxide on cells.
v If found to be not toxic, potential applications of carbon nanomaterials
include the use of graphene scaffolds for nerve tissue repair and tissue
engineering (Dvir et al. 2010).
Add coverslips
to 6 well
plates
Stain used:
Anti-NeuN, anti-MAP2,
anti-βIII Tubulin, anti-LAMP1, anti-
NeuroD1, Phalloidin, DAPI or Giemsa
May-Grundwald
Neuro-2A BEAS-2B
Techniques employed:
v Cell culture
v Giemsa staining
v Light microscopy
v Live imaging
v Fluorescence microscopy
v Immunocytochemistry
A549
Seed with Neuro-2A
cells in MEM Eagle
Seed with BEAS-2B cells
in RPMI
Seed with A549 cells in
F-12 Nutrient Mixture
Ham
DAPI
Neuro-D1
MergeAntibody
-veControlβIII-
Tubulin
Antibody
LAMP1-veControl
Phalloidin
v Phalloidin staining of Neuro2A cells
DAPI
Phalloidin DAPI Merge
Fix cells using
MeOH or 4%PFA
Alturkistani, H. A., et al. (2016). Histological Stains: A Literature Review and Case Study. Global Journal of Health Science, 8(3), 72–79.
Dvir, T., et al. (2011). Nanotechnological strategies for engineering complex tissues. Nature Nanotechnology. 1(6), 13-22.
Musumeci, G. (2014). Past, present and future: overview on histology and histopathology. Journal of Histology and Histopathology, 1(1), 5. Wulf, E.,
Deboben, a, Bautz, F. et al. (1979). Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proceedings of the National Academy of Sciences of
the United States of America, 76(9), 4498–4502.
EXPERIMENTAL
NeurN