0115 966 7955 Today's Opening Times 10:00 - 20:00 (BST)

Developing Visual Data on PCNA Markers

Published: Last Edited:

Disclaimer: This essay has been submitted by a student. This is not an example of the work written by our professional essay writers. You can view samples of our professional work here.

Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of UK Essays.

Detection of proliferating cell population in intestinal, liver and brain tissues by way of proliferating cell nuclear antigen (PCNA) and use of diaminobenzidine (DAB).

  • Lewis Buchan

Introduction and Aims

Immunohistochemistry as with immunocytochemistry is a visual aid in the clinical or research examination of cell growth, disease and therapy response by variation in cell protein changes (Hannon-Fletcher and Maxwell, 2009). Identification of proliferating cells without use of live animal injection of anti-BrdU can be achieved by the less complex yet established method of PCNA detection (Muskhelishvili et al., 2003). All proliferating cells express proliferating cell nuclear antigen, known as PCNA (DNA polymerase δ auxiliary protein) in the cell nucleus. It is common practise to monitor DNA synthesis with the use of PCNA as a marker (Ku et al., 1989). Immunohistochemistry (IHC) and PCNA colour development is often used to establish tissue cell proliferation; whether the tissue is in a state of normality or if visible detection of colour development is established; in a mitotic state or perhaps repair growth. Neoplasia or dysplasia was not considered in this report. Cell proliferation detection takes advantage of the immune response to specific antigens. Tissues of different organs will mostly regenerate during times of growth which can be mature, infantile or even in the foetus (Iatropoulos and Williams, 1996). Types of growth that the following practical is interested in are proliferation in mature mice tissues rather than differentiation. Growth and proliferation of mature cells can be due to spontaneous growth by regular cell cycles. This is considered true of labile and stable tissue but not for everlasting tissue cells (Iatropoulos and Williams, 1996). The lab practical was set up to establish and evaluate three mouse tissue types in relation to the labile (renewing), stable (expanding) and everlasting (static) classification of Bizzozero in 1894 (Bizzozero, 1894).

The aims of the practical were to further acquire basic visual data on PCNA markers (by way of diaminobenzidine tablets, (DAB)) of the three tissue samples to ascertain which of the three (brain, gut or liver) has greater or less proliferation in comparison to each other and the negative control. The introduction of the primary antibodies would in theory bind to the antigen’s epitopes.

Methods and Materials

Blocking with normal serum was applied for 30 minutes before the primary antibody is introduced so that cell proteins other than proliferating cell populations are not marked by the staining. Prepared antibodies are introduced to amplify individual cells that are regenerating. Two non-specific polyclonal anti mouse PCNA reagents were required for establishing a cross reaction with the antigens on the tissue samples. The antibodies are 2426 primary rabbit anti mouse antibody and 6721 secondary goat anti rabbit, HRP conjugated. The use of two different antibodies amplifies the antigen’s signal as displayed in figure 1. The secondary antibody is raised against the primary which was raised against the PCNA. An indirect, two step process that doubles the signal for microscopy viewing.

C:\Users\Owner\Desktop\IHC.LabReport\Antibody.HRP.DAB.jpg

Figure 1. Graphic version of primary and secondary antibody attachment to the mouse antigen (indirect). Enzyme (HRP)/antibody complex with DAB substrate development (Abcam.com, n.d.).

Sigmafast 3,3’ – diaminobenzidine tablets were used as a chromogen. The sample tissue slides A, B, C and a control were prepared by washing 2 x 5 min in TBS plus 0.025% Triton X-100, then blocked in 10% normal serum with 1% BSAin TBS for 30 mins at room temperature. Then, working specifically with sample B only, we continued preparation by applying 80µl of the primary antibody; polyclonal Rabbit anti-mouse PCNA antibody (diluted 1: 500 in TBS with 1% BSA), and incubated for 45 mins at 37°C. Sample B was then rinsed 2 x 5 min with TBS 0.025% Triton in a coplin jar and transferred to 0.3% H2O2in TBS and incubated at room temperature for 10 min in a coplin jar. The slide was then rinsed three times with water. 80µl of secondary antibody; goat anti-rabbit, coupled with Horse radish peroxidase (HRP), diluted 1 in 1000 in TBS with 1% BSA, was applied and incubated for 30 mins at 37 oC.The slide was then washed 3 x 5 mins with TBS+1% BSA. Chromogen, (300µl of DAB peroxidase (Sigma D1448)) was then applied to develop for 10 min at room temperature. The tissue slide was further rinsed for 5 min before introducing haematoxylin as a counterstain for 2 mins. The slide were then subjected to gentle rinsing until a blue appearance was introduce on the tissue to give non PCNA stained cells some colour. Slides were viewed by brightfield microscopy under various magnitudes of 10x and 20x objectives. Photographs were achieved by simply using a smartphone camera with the lens placed within 1cm of the microscope eyepiece.

Results

In the first instance, identification of the three tissue samples A, B and C was as follows: sample tissue A was identified as gut, sample B was identified as liver and sample C was identified as brain tissue; all shown in figure 2. Samples A and C were prepared by various teams while tissue B was prepared (and photographed) specifically by the author’s team. Although tissue B is taken with a 10x objective it was not established how the other tissues were magnified and photographed.

IHC-A-Gut.jpg IHC-3.jpg

IHC-C-Brain.jpg

Figure 2. Immunohistochemical staining of sample mouse tissues: [A] Gut tissue shows strong proliferation (H & E as counterstain), [B] Liver tissue is showing spots of proliferation (H & E as counterstain) and [C] Brain tissue showing no proliferation (methylene blue as counterstain).

All images are then cropped for visual purposes. This means size relationship between the three tissues is not in exact proportion to each other. Initial observations were slightly confusing when looking at only one slide sample. In our case sample B was difficult to read to an untrained eye. It was initially unclear which areas of tissues had more DAB attached to PCNA cells, i.e. in a state of proliferation. Observation of the three different tissues demonstrates three distinct degrees of staining from the DAB. Tissue A (gut) had the most prolific and obvious staining; presenting a high account of cell proliferation. Tissue B (liver) has only a few cells of staining. Some of those cells are indicated by arrows. This represents a low account of proliferation; especially in contrast to tissue A. Tissue C (brain) had no real obvious signs of cell proliferation staining. There is however extensive methylene blue attachment that is perhaps the fatty acid components of myelin sheath. The negative control was set up to not show colouring from the chromogen (not shown due to errors). Observations of the tissues were initially viewed under a 10x objective to find an area were staining had taken effect. The objective lens was occasionally increased to 40x once proliferation areas were established to enhance examination detail (no 20x objective on the microscope hindered examination). If PCNA is a way to observe cell division and to establish classification of the tissues in accordance with Bizzozero’s system then the results for the tissues confirm three clear separation of categories know as type 1: labile, type 2: stable and type 3: perennial/everlasting; as shown in table 1.

Tissue

Labile

(Renewing or proliferating)

Stable (Expanding)

Perennial/Everlasting (static or non-proliferating)

A - Gut

Positive

Negative

Negative

B - Liver

Negative

Positive

Negative

C - Brain

Negative

Negative

Positive

Control

Negative

Negative

Negative

Table 1. Presenting observed results of A, B & C tissues in accordance with Bizzozero categorisation. Positive: correlates with tissue type. Negative: does not correlate with tissue type.

Samples A and B were counterstained with haematoxylin while sample C was counterstain with methylene blue. Both counterstaining methods gave contrast to the brown of DAB. It appears the methylene blue in sample C has left large spots of residue while not showing any proliferation.

Discussion

There were plans to assess if the IHC staining methodology worked in comparison to the negative control tissues. However the control tissues were not available. The process of looking at three various tissues from three mouse organs was initially concerned with clarification of tissues types by cell proliferation; to distinguish the unknowns of A, B or C, which were liver, brain or intestine. Consideration was also given to comparison of propensity for cell proliferation in the three tissue samples. Cells die and need replaced. Healing, hyperplasia and spontaneous growth of tissues due to injury or general maintenance of tissue health is part of the regeneration that is being observed here. The cell proliferation was exploited by taking advantage of antigen, antibody specificity and use of a visible marker substrate of an enzyme conjugated to a secondary antibody complex. An often used alternative to immunohistochemistry is immunofluorescence. Fluorescent dyes deteriorate with time and requires UV modified microscopy (Cross, Underwood and Underwood, 2013). It does not seem necessary in this instance as the opaque deposits of DAB were sufficient. Moreover for the purposes of this experiment there seems no particular reason to apply more than the two antibodies to amplify proliferating cells.

Degrees of proliferation in the samples were clearly evident once adjustment to the detection method was perfected. There was additional time outside of the lab to analyse the photographic images. It became quite clear when there was PCNA marking and when there was not. There are distinct areas of the tissue in samples A (gut) and individual spots in B (liver) where cell division was clearly seen. It was assumed this was due to natural regeneration of tissue perhaps stimulated by spontaneous growth. In plain terms growth factors released during G1 phase of the cell cycle stimulate proliferation (Iatropoulos and Williams, 1996). Expression of PCNA (a 36 kD nonhistone nuclear protein) in the cell nuclei is considered to occur as part of DNA synthesis in S phase. More specifically from late G1 through S and G2 and into early M phase (Iatropoulos and Williams, 1996). Observation of PCNA is therefore a signal of mitotic cells. Quantities of this signalling can be indexed into the Bizzozero categories and therefore a tissue’s regeneration capabilities. Samples A, B and C can be attributed to the three biological category states of labile, stable and perennial respectively. Bizzozero did consider which tissue types fitted into his categories; clearly set out in table 2.

Category

Tissue examples

Labile (Renewing)

Gut, gastric, epithelial coverings, ovary, testicle, lymph, sebaceous, bone marrow and spleen.

Stable (Expanding)

Liver, kidney, pancreas, bones, smooth muscles and salivary glands.

Perennial (Everlasting)

Nervous and striated muscle

Table 2. Categorisation of tissues in line with Bizzozero (Bizzozero, 1894) (Mazzarello et al., 2001).

The gut sample A offers clear areas of proliferation. This is unsurprising as research of intestinal epithelial cells in mice or rats is wide spread. The small intestine alone has a mitotic cell population in abundance. Human epidermal growth factor stimulates epithelial cell proliferation (Despopoulos and Silbernagl, 1991) and keratinocyte growth factor supports crypt cell proliferation while suppressing epithelial apoptosis (Cai et al., 2012). Furthermore, crypts of Lieberkuhn cells replenish the villi tips creating intestinal cell regeneration on a continuous cycle (Despopoulos and Silbernagl, 1991). The liver sample B has a small quantity of what looks like individual cells in a state of proliferation. This compares well with evidence describing the liver of mice having good regeneration abilities of hepatocytes and parenchymal cells (Cressman et al., 1996). Regeneration of liver cells after tissue loss or toxicity damage is thought in part to rely upon STAT3 DNA stimulation from cytokines, interluken-6 and epidermal growth factor (Cressman et al., 1996). Without IL-6, proliferation is greatly diminished in hepatocyte regeneration and causes irregularity in the G1 phase of the cell cycle according to Cressman et al. There is no appearance of proliferation occurring in the results from the brain tissue in sample C. This again correlates with Bizzozero and the proposition by Ramon y Cajal that cessation of nerve expansion in postnatal mammals is expected (Colucci-D’Amato, Bonavita and di Porzio, 2006). Up until 1960 it was considered from the established “dogma” of Bizzozero and later Ramón y Cajal that neurogenesis in adult vertebrates did not occur (Bottenstein, 2003). Gradually new techniques for brain cell proliferation using tritium labelled to DNA thymidine began dispute this assumption. Increasing evidence points towards vertebrates’ potential to create fresh neurons during experimental trails (Raucci et al., 2006). The formation of neural stem cells and neurogenesis has now been established and is part of research into neurological conditions (Colucci-D’Amato, Bonavita and di Porzio, 2006).

Conclusions

From the results it can be seen that disparate tissues proliferate at dissimilar rates. The method of proliferation detection utilised takes a degree of skill to prepare and interpret. It would have been useful to have known the mouse age of the tissue samples and if there were any known health issues. It was assumed that the animals were free from ill health and fully mature, i.e. there was no developmental growth in the brain as can occur with young mammals. The tissue types once identified, comfortably fitted into the categories as outlined by Bizzozero. However, it was considered his original classification needs some updating in parts. More recent discoveries show the potential of nerve cells to divide and ultimately regenerate as outlined in the discussion.

Improvement on visual clarity could potentially have been improved if a higher magnification objective was used to scrutinise individual cells and perhaps to have observed which parts of the cell the chromogenic staining took place. It is assumed the use of 100x, or even 200x objectives would allow possible identification of the nuclei in some cell types. Perhaps observing which part of the cell or nucleus has actually taken up the stain. This would have given more data to cross reference with previous studies.

Due to high regularity of cell proliferation in mice intestinal tissues undertaken in laboratory experiments it makes good sense that these specific tissues are the ideal material to use in biological testing for exogenous or endogenous factors affecting the activation or suppression of abnormal growth. Taken as a whole the experiments were informative and gave a good understanding of the methods. Further experiments to broaden perspective and analysis would be to look at tissues with abnormal growth in the form of tumours; to further expand on the visual significance of cell and tissue irregularities.

References

Abcam.com, (n.d.). Antibody, HRP, DAB. [image] Available at: http://www.abcam.com/index.html?pageconfig=resource&rid=12865&source=pagetrap&viapagetrap=expose [Accessed 23 Feb. 2015].

Bizzozero, G. (1894). An Address on the Growth and Regeneration of the Organism: Delivered before a General Meeting of the XIth Internationa Medical Congress, Held in Rome, 1894. BMJ, 1(1736), pp.728-732.

Bottenstein, J. (2003). Neural stem cells. Boston: Kluwer Academic Publishers, p.182.

Cai, Y., Wang, W., Liang, H., Sun, L., Teitelbaum, D. and Yang, H. (2012). Keratinocyte Growth Factor Improves Epithelial Structure and Function in a Mouse Model of Intestinal Ischemia/Reperfusion. PLoS ONE, 7(9), p.e44772.

Colucci-D’Amato, L., Bonavita, V. and di Porzio, U. (2006). The end of the central dogma of neurobiology: stem cells and neurogenesis in adult CNS. Neurological Sciences, 27(4), pp.266-270.

Cressman, D., Greenbaum, L., DeAngelis, R., Ciliberto, G., Furth, E., Poli, V. and Taub, R. (1996). Liver Failure and Defective Hepatocyte Regeneration in Interleukin-6-Deficient Mice. Science, 274(5291), pp.1379-1383.

Cross, S., Underwood, J. and Underwood, J. (2013). Underwood's pathology. Edinburgh: Churchill Livingstone.

Despopoulos, A. and Silbernagl, S. (1991). Color atlas of physiology. Stuttgart: G. Thieme.

Goers, J. (1993). Immunochemical techniques laboratory manual. London: Academic Press.

Hannon-Fletcher, M. and Maxwell, P. (2009). Advanced techniques in diagnostic cellular pathology. Chichester, West Sussex: John Wiley & Sons.

Iatropoulos, M. and Williams, G. (1996). Proliferation markers. Experimental and Toxicologic Pathology, 48(2-3), pp.175-181.

Ku, D., Travali, S., Calabretta, B., Baserga, R., Ottavio, L. and Rizzo, M. (1989). Structure of the human gene for the proliferating cell nuclear antigen. J. Biol. Chem., 264(13), pp.7466-7472.

Mazzarello, P., Mazzarello, P., Calligaro, A. and Calligaro, A. (2001). TIMELINE: Giulio Bizzozero: a pioneer of cell biology. Nature Reviews Molecular Cell Biology, 2(10), pp.776-784.

Muskhelishvili, L., Latendresse, J., Kodell, R. and Henderson, E. (2003). Evaluation of Cell Proliferation in Rat Tissues with BrdU, PCNA, Ki-67(MIB-5) Immunohistochemistry and In Situ Hybridization for Histone mRNA. Journal of Histochemistry & Cytochemistry, 51(12), pp.1681-1688.

Polak, J. and Van Noorden, S. (1997). Introduction to immunocytochemistry. Oxford, OX, UK: BIOS Scientific Publishers.

Raucci, F., Di Fiore, M., Pinelli, C., D’Aniello, B., Luongo, L., Polese, G. and Rastogi, R. (2006). Proliferative activity in the frog brain: A PCNA-immunohistochemistry analysis. Journal of Chemical Neuroanatomy, 32(2-4), pp.127-142.

  • Lewis Buchan

To export a reference to this article please select a referencing stye below:

Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.

Request Removal

If you are the original writer of this essay and no longer wish to have the essay published on the UK Essays website then please click on the link below to request removal:


More from UK Essays

We can help with your essay
Find out more
Build Time: 0.0050 Seconds