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Chapter 3. General Methods Human Tissue Processing
Post-mortem tissue processing
The human post-mortem brain tissue used for this project was obtained from the Neurological Foundation of New Zealand Douglas Human Brain Bank at the University of Auckland. The tissue was donated to the Brain Bank with family consent and its use for these studies was approved by the University of Auckland Human Participants Ethics Committee. The control cases had no history of cognitive impairment and cause of death was unrelated to any neurological condition. Control cases selected for this study had a maximum Braak stage of II as determined by independent pathological analysis, corresponding to β-amyloid plaque and tau NFT changes consistent with that of normal aging (Braak and Braak, 1995). The AD cases selected for this study had a minimum Braak stage of IV and a clinical history of dementia with no evidence of other neuropathology.
Fresh frozen tissue processing
The post-mortem brain processing protocol has been published in detail by Waldvogel et al., 2007. Fresh frozen tissue was used for western blotting as fixation has been shown to reduce the effectiveness of protein extraction. Donated brains were obtained at autopsy and the left hemisphere was immediately dissected into blocks representing functional regions of the brain (Waldvogel et al., 2007). These blocks were frozen using CO2 powder, wrapped in tinfoil and stored at -80°C. For western blotting the frozen blocks were cut into 30-µm sections and the region of interest was scraped from the slide using a razor blade and collected in an Eppendorf tube. This tissue was stored at -80°C until protein extraction was performed.
Formalin-fixed frozen tissue processing
Formalin-fixed frozen tissue was used for free-floating immunohistochemistry staining as fixation preserves tissue integrity and cellular morphology. The right hemisphere of each brain was processed in this manner. Phosphate-buffered saline (PBS) containing 1% sodium nitrite was first perfused through the cerebral arteries to dilate the vasculature and clear blood from the brain. The hemisphere was then perfused with 15% formalin in 0.1 M phosphate buffer and then immersed in this same solution overnight. Following fixation, the brain was dissected into functional blocks in the same manner as the fresh hemisphere. These fixed blocks were subsequently cryoprotected by immersion in 20% sucrose for one week and 30% sucrose for three weeks before being frozen
using CO2 powder, wrapped in tinfoil and stored at -80°C. For free-floating immunohistochemical staining, 50-µm-thick coronal sections were obtained from each block using a freezing sliding microtome (Microm, HM450) and stored at 4°C in PBS containing 0.1% sodium azide. Solutions for post-mortem tissue processing are outlined in Table 3.
Paraffin-embedded formalin-fixed tissue processing
Paraffin-embedded formalin-fixed tissue was used for the independent pathological assessment of each case and also for the construction of tissue microarrays. During dissection of the formalin-fixed hemisphere, 1-cm-thick tissue blocks were taken to be embedded in paraffin (detailed by Waldvogel et al., 2007). This processing was carried out by the Histology Laboratory at the University of Auckland, School of Medical Sciences. Briefly, tissue blocks were dehydrated in a series of ethanol solutions, cleared in xylene and embedded in paraffin wax. For immunohistochemistry, paraffin-embedded tissue was cut into 7-µm-thick sections using a rotary microtome (Leica Biosystems RM2235) and mounted onto Superfrost-Plus charged slides (Menzel-Glaser) using a 37 °C water bath (Leica Biosystems, H1210).
Construction of tissue microarray
Tissue microarrays allow the analysis of small tissue samples from multiple cases on a single section. The SVZ tissue microarrays used in this study were prepared by Claire Lill (Research technician, CBR, University of Auckland) using paraffin embedded formalin-fixed SVZ samples obtained from control and AD cases. As illustrated in Figure 3.1, 2 mm diameter cores were extracted from the dorsal, middle, and ventral aspects of the SVZ and inserted into a recipient paraffin block. Sections of 7 µm thickness were cut using a microtome and mounted onto slides in preparation for immunoperoxidase staining.
Immunohistochemistry
Immunoperoxidase Staining of Free-Floating Tissue
Immunoperoxidase staining uses antibodies in conjunction with a peroxidase enzyme to label a protein of interest with a stable chromogenic product that can be visualised using brightfield microscopy (Figure 3.2A). Sections to be stained in this manner were first treated with the appropriate antigen retrieval procedure as optimised for each antibody (Table 4). Endogenous peroxidase activity was then blocked by immersion in a solution of 50% methanol and 1% H2O2 for 20 minutes at room temperature (RT). The sections were incubated in primary antibody for 72 hours on a rocking platform at 4°C. A biotinylated secondary antibody specific to the primary antibody species was then applied for 24 hours at RT. This was followed by ExtrAvidin®-Peroxidase (E2886, Sigma; diluted 1: 1,000) for 4 hours at RT. Finally, a solution of 3,3-diaminobenzidine chromogen (DAB) in 0.1 M phosphate buffer and 0.01% H2O2 was used to produce a stable brown stain. In some instances, 0.04% Nickel Ammonium Sulfite was added to the solution to produce a black stain rather than a brown stain for greater contrast (section 7.3.1). Primary antibodies, secondary antibodies and ExtrAvidin®-Peroxidase were diluted in a blocking buffer of PBS containing 1% sera from the species in which the secondary antibody was raised and 0.04% merthiolate. Sections were washed in PBS-T (3 x 10 minutes) between each step. The sections were mounted onto glass slides using a 0.5% gelatin solution and left to dry for two days prior to being dehydrated in an ascending series of alcohol (75% to 100%) then cleared in xylene. Coverslips were applied using DPX mounting media (06522, Sigma). Solutions for immunohistochemistry are outlined in Table 7.
Cresyl violet counterstain
Cresyl violet labels the nissl bodies present in cells and was used in this study as a counterstain to delineate regions of interest. The cresyl violet procedure was carried out in conjunction with immunoperoxidase staining as well as independently on unstained sections mounted on glass sides using 0.5% gelatin. Sections were dried at RT for two days prior to staining. Once dry, the sections were first washed in distilled H2O for 5 minutes before being dehydrated in an ascending series of alcohol to xylene (5 minutes each in 75%, 85% and 95% ethanol, 3 x 10 minutes in 100% ethanol, and 10 minutes in xylene). The xylene solution turned cloudy if the sections were not adequately dehydrated. In this situation, the sections were returned to a fresh solution of 100% ethanol before being returned to xylene. Once cleared, the sections were rehydrated through the same alcohol series in reverse order. After a 5-minute wash in distilled H2O, the sections were immersed in cresyl violet stain for 15 minutes. Sections were then dehydrated and coverslipped as described above in section 3.2.1.
Chapter 1. General Introduction
Chapter 2. Literature Review
2.1 Alzheimer’s disease
2.2 Human neurogenesis
2.3 The role of PSA-NCAM in neurogenesis and structural plasticity
2.4 Aims of this thesis
Chapter 3. General Methods
3.1 Human Tissue Processing
3.2 Immunohistochemistry
3.3 Golgi Staining .
3.4 Western Blotting
Chapter 4. Cell Proliferation and Neurogenesis in the Alzheimer’s Disease Hippocampus 49
4.1 Introduction
4.2 Methods Overview
4.3 Results
4.4 Discussion
4.5 Summary
Chapter 5. Cell Proliferation in the Alzheimer’s Disease Sub-Ventricular Zone
5.1 Introduction
5.2 Methods
5.3 Results
5.4 Discussion
5.5 Summary
Chapter 6. Distribution of PSA-NCAM in Normal, Alzheimer’s and Parkinson’s Disease
Human Brain
6.1 Introduction6.2 Methods
6.3 Results
6.4 Discussion
6.5 Summary
Chapter 7. Neurochemical Characterisation of PSA-NCAM+ cells in the Normal and Alzheimer’s Disease Human Brain
7.1 Introduction
7.2 Methods
7.3 Results
7.4 Discussion
7.5 Summary
Chapter 8. General Discussion
8.1 Neurogenesis in the Alzheimer’s disease brain
8.2 PSA-NCAM-mediated plasticity in the Alzheimer’s disease brain
8.3 Concluding Remarks
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Plasticity in the Human Alzheimer’s Disease Brain
