Optimizing Immunohistochemical Neuropathological Protocols for Human Brain Banking

Abstract

Background: The neuropathological diagnosis in post-mortem human brain is the golden standard and crucial for achieving the ultimate goal of serving brain research via human brain banking. With the construction of a China brain bank network, it is urgent to establish a unified protocol, including standardized neuropathological staining protocols, to ensure identical and reliable diagnostic results across different brain banks and to enable future sample sharing for research.

Method: This study focused on optimizing immunohistochemical (IHC) staining protocols for four key neuropathological markers of neurodegenerative diseases: hyperphosphorylated Tau protein, β-amyloid, α-synuclein, and phosphorylated transactive response DNA-binding protein 43. The optimization was performed on formalin-fixed, paraffin-embedded human brain tissues by modifying conditions including antigen retrieval. The optimized stainings were compared with those from the highly standardized Netherlands Brain Bank.

Results: Through protocol optimization and comparison with the Netherlands Brain Bank standards, we obtained optimal and consistent staining results suitable for reliable neuropathological diagnosis.

Discussion: This study provides a solid foundation for quality control of IHC staining and for accurate neuropathological diagnosis within the framework of China brain banking.

Graphical abstract

Keywords

human brain banking / immunohistochemistry / neuropathological diagnosis

Introduction

A human brain bank is a scientific infrastructure that provides the research community with high-quality, clinically and neuropathologically well-characterized, and neuroanatomically well-defined postmortem brain samples of brain disorders and their appropriately matched controls, with the aim of promoting research on the nature of human brains and on the pathogenesis of neuropsychiatric diseases[1]. For such a purpose, it is of key importance to set up a standardized operational protocol (SOP) for human brain banking, which covers the whole procedure from brain sample collecting within the ethic and professional request, sample processing and storing, neuropathological diagnosis, and sample dispatching. Establishing the correct neuropathological diagnosis of each case is of key importance for subsequent post-mortem research. It is not only needed to indicate the stage of the disease, such as the Braak stages for Alzheimer’s disease[2,3] or for Parkinson’s disease[4], but also it may revise or add to the antemortem/clinical diagnoses in about 25% of the cases[5]. Neuropathological diagnoses are established based upon a series of brain section stainings including hematoxylin-eosin (H&E) staining, histochemical stainings, and immunohistochemistry (IHC) stainings for specific pathological proteins that are characteristic for different diseases. The stainings are applied according to brain banking SOP on sections of the following brain areas: the hippocampus, amygdala, frontal lobe, temporal lobe, occipital lobe, parietal lobe, basal nucleus, substantia nigra, medulla oblongata, and cerebellum. It is worth noting that although H&E staining can distinguish cellular structures and cell types by contrasting nuclear and cytoplasmic staining, it cannot trustfully distinguish neuropathological changes. On the other hand, although histochemical staining such as Haga methenamine silver (M-Ag), Gallyas, and Kluver-Barrera staining may detect special pathological changes, such as amyloid plaques, neurofibrillary tangles, and (loss of) myelin, they hold disadvantages including lower sensitivity, and thus missing the early changes of disease markers[3]. Therefore, the more sensitive IHC staining including those for the hyperphosphorylated Tau (p-Tau), α-synuclein (α-Syn), β-amyloid (Aβ), and phosphorylated transactive response DNA-binding protein 43 (p-TDP43) are crucial for the diagnosis, especially the diagnosis of neurodegenerative diseases that are frequently seen in brain banking[2,6–8]. These IHC results are, however, significantly affected not only by the source of the antibodies, but also by the staining protocols from the section pretreatment to signal detection conditions, all of which may lead to discrepancies in the results among different brain banks[8]. At present, although China Human Brain Bank Consortium has published the SOP for brain tissue collection[9], a SOP for such staining protocols is lacking. For the purpose of standardization of the neuropathological diagnoses among different brain banks, it is crucial to establish standardized IHC staining protocols for the four IHC stainings mentioned above, based upon the positive control staining on sections obtained from a well-established human brain bank.

The present study was therefore conducted to optimize the IHC staining protocols for p-Tau, α-Syn, Aβ and p-TDP43, using human brain tissue samples collected by China Brain Bank of Zhejiang University (CBB-ZJU), i.e., the National Health and Disease Human Brain Tissue Resource Center. The optimized IHC staining protocols were compared with those used by the Netherlands Brain Bank (NBB) which is a global leader brain bank in open access, clinical documentation, and ethical aspects[10]. In addition, the protocols were used on human brain tissue samples obtained from NBB to verify the reliability.

Materials and methods

Post-mortem human brain material

Post-mortem human brain tissues were obtained from CBB-ZJU and NBB following permission for brain autopsy and the use of the brain tissues and clinical data for research purposes[5,11]. Cases with AD, Parkinson’s disease, limbic-predominant age-related TDP-43 encephalopathy (LATE), or comorbidity of them were selected for the present studies (Table 1).

In both CBB-ZJU and NBB, during autopsy, one hemisphere of the brain was immediately fixed in 10% formalin solution. After 4 weeks of fixation, this hemisphere was dissected according to the SOP to obtain tissue samples from different brain regions for stainings and diagnosis (for details of the brain regions see[9]). For the present study, sections of hippocampus, amygdala, middle frontal cortex, parietal cortex, and the upper medulla oblongata (containing the X and Ⅻ cranial nerve nuclei, as well as the inferior olivary nucleus) were used. Tissues were placed in tissue cassettes, dehydrated in gradient ethanol series, cleared in xylene, and infiltrated with paraffin wax to displace xylene. The tissues were then embedded in paraffin wax. The paraffin-embedded tissue blocks from CBB-ZJU were then cut into serial sections of 6-µm thickness, the same as sections from NBB, with a microtome (Leica RM2235, Germany).

IHC experiment

Original IHC staining protocols used in CBB-ZJU for p-Tau, α-Syn, Aβ, and p-TDP43

Originally, the IHC protocols for p-Tau, α-Syn, Aβ, and p-TDP43 were as the follows:

i) The sections were deparaffinized and rehydrated following using xylene (100234192, China Pharmaceutical Group Corporation) and decreasing gradient of ethanol (100092680, China Pharmaceutical Group Corporation). Subsequently the sections were rinsed in distilled water for 3–5 s and then put in PBS solution (pH = 7.3, 3 × 5 min, ZLI-9061, Zhongshan Jinqiao).

ii) Then the sections were put in 1% H₂O₂ (10011218, China Pharmaceutical Group Corporation) for 15 min and rinsed in the distilled water for 3–5 s to eliminate endogenous peroxidase.

iii) For antigen retrieval, the sections were brought to boiling citric acid buffer (pH = 6.0, PM5100, Coolaber) for 15 min, cooled to room temperature (RT), washed with PBS solution (pH = 7.3, 3 × 5 min). For α-Syn staining, the sections were put in 80% formic acid (10010118, China Pharmaceutical Group Corporation) for another 15 min at RT, then washed with PBS solution (pH = 7.3, 3 × 5 min).

iv) The sections were incubated with the respective primary antibody diluted in PBS solution containing 10% goat serum (SL038, Solarbio) and 0.01% Triton X-100 (SLB73016, Sigma) at 4°C overnight in humidity chamber. For p-Tau staining, the sections were first incubated at 37°C for 1 h in the humidity chamber before moved to 4°C for overnight. Note the working dilutions are 1:100 (p-Tau), 1:100 (Aβ), 1:50 (α-Syn), and 1:300 (p-TDP43), respectively. Primary antibodies used were commercially available and shown in Table 2.

v) The sections were incubated with the primary antibody in 37°C thermostat for 1 h. The sections were washed with PBS solution (pH = 7.3, 3 × 5 min) and incubated with respective secondary antibodies according to instructions of the kits (Table 2) for 1 h at 37°C thermostat.

vi) The sections were washed with PBS solution (pH = 7.3, 3 × 5 min). Sections were then incubated with DAB (3,3’-diaminobenzidine tetrahydrochloride) solution kit (ZLI-9018, ZSGB-BIO) until the brown protein sediment was found under the microscope.

vii) The sections were washed with PBS solution (pH = 7.3, 3 × 5 min) and then putted in hematoxylin solution (G1150, Solarbio) for 3 min.

viii) The sections were placed in slowly running tap water until the water became clear. They were then passed through an increasing gradient of ethanol for dehydration and through xylenes for transparency in an automatic staining machine (Leica Autostainer X1, Germany), and subsequently cover-slipped with neutral resin (10004160, China Pharmaceutical Group Corporation) using an automatic cover-slipping machine (Leica CV5030, Germany).

Optimization of IHC staining protocols for p-Tau, α-Syn, Aβ, and p-TDP43

Because of the suboptimal staining results obtained using the original IHC protocols of CBB-ZJU (see Results section), we modified conditions in the protocols to optimize the results as described below:

Adjustment of the pH of antigen retrieval solution

IHC staining for p-Tau (AT8), α-Syn (KM51), Aβ (BAM-10), and p-TDP43 (p-Ser 409/410) were performed on the sections of the hippocampus, parietal cortex, and amygdala. The boiling Tris-EDTA antigen retrieval buffer (pH = 9.0, IH0297, Leagene) was compared with the boiling citric acid buffer (pH = 6.0, as used in the original protocol), with both applied for 15 min, with the rest steps of protocol remained consistent with those described in post-mortem human brain material.

For p-Tau and α-Syn staining, Tris-EDTA buffer (pH = 9.0) yielded better results than with citric acid buffer (pH = 6.0). We tried to further optimize the staining protocol for α-Syn to reduce the staining time and further improve the staining results. For Aβ staining, Tris-EDTA buffer (pH = 9.0) did not show significant improvement for Aβ signal and the background remained high. Moreover, neither Tris-EDTA buffer (pH = 9.0) nor citric acid buffer (pH = 6.0) showed ideal staining for p-TDP43 staining, reflected as a non-specific nuclear staining and/or over-stained background. See details in the Results section. Thus, further optimization of their respective staining protocols was needed.

Further optimization of IHC staining protocol for α-Syn

Based on these original protocols, three antigen retrieval conditions for α-Syn staining were compared in the medulla oblongata sections as follows: i) boiling in Tris-EDTA buffer (pH = 9.0, 15 min); ii) incubation in 80% formic acid (15 min, RT); and iii) sequential treatment with boiling Tris-EDTA buffer (pH = 9.0, 15 min) followed by 80% formic acid (15 min, RT). The rest steps were the same as those used for the post-mortem human brain material. Additionally, for condition iii, different treatment times were compared: boiling in Tris-EDTA buffer (pH = 9.0) for 15, 20, or 30 min, followed by incubation in 80% formic acid at RT for 10 or 15 min.

Further optimization of IHC staining protocol for Aβ

Given the use of multiple Aβ antibodies including BAM-10, 6E10, and 6F3D across different CBB labs, we started the optimization with BAM-10 staining on the parietal cortex sections. Based on these original protocols, two antigen retrieval conditions were compared: i) boiling citric acid buffer (pH = 6.0, 15 min); ii) boiling citric acid buffer (pH = 6.0, 15 min) followed by 80% formic acid (10 min, RT). The rest steps were the same as those used for the post-mortem human brain material.

After the optimization, we stained parietal or middle frontal cortex sections using BAM-10, 6E10, and 6F3D as primary antibodies to compare staining efficiency.

Further optimization of IHC staining protocol for p-TDP43

Based on these original protocols, citric acid buffer (pH = 6.0, 15 min) was used for antigen retrieval of p-TDP43 staining. To determine the optimal primary antibody dilution, frontal cortex sections were stained using dilutions of 1:1000, 1:2000, 1:3000, and 1:4000.

Validation of optimized IHC staining protocols

To validate the optimized CBB-ZJU IHC staining protocols for p-Tau, Aβ, α-Syn, and p-TDP43 (Table 3), tissue sections obtained from NBB and CBB-ZJU (Table 1) were used respectively, among which the hippocampus sections were used for p-Tau and Aβ staining, and amygdala sections for α-Syn and p-TDP43 staining.

NBB sections were stained using the optimized CBB-ZJU IHC protocols corresponding to the four respective antibodies. Each antibody was tested in duplicates. Parallel staining was also performed in the NBB laboratory with NBB standard IHC protocols (Table 3) on the same batch of NBB sections. In addition, CBB-ZJU sections were stained in NBB laboratory using NBB IHC protocol, and the same batch of CBB-ZJU sections was also stained in the CBB-ZJU laboratory using the optimized CBB-ZJU protocols. The staining results from the two laboratories were compared to assess inter-laboratory consistency and the reliability of the CBB-ZJU optimized protocols. Staining in each laboratory was conducted by a technician who was blinded to the sample origins.

Image analysis

All stained sections were scanned using a MAGSCANNER KF-PRO-002 scanner (Jiangfeng Bioinformatics, China), and digital images were captured with K-Viewer software (1.7.1.1). The images were then reviewed by a neuropathologist who was blinded to the IHC staining protocols for diagnostic evaluation.

Results

Original IHC staining protocols used in CBB-ZJU for p-Tau, α-Syn, Aβ, and p-TDP43

Using the original IHC staining protocols on CBB-ZJU sections (Table 1), for p-Tau and α-Syn staining, the positive signals were in general too weak (Figure 1A and B), and for Aβ and p-TDP43 staining, the signals were too weak and with background over-staining (Figure 1C and D). In addition, some non-specific nuclear p-TDP43 staining was observed (Figure 1D).

Optimization of IHC staining protocols for p-Tau, α-Syn, Aβ, and p-TDP43

Adjustment of the pH of antigen retrieval solution

Compared with the original protocol using citric acid buffer (pH = 6.0) for antigen retrieval of p-Tau, α-Syn, Aβ, and p-TDP43 (Figure 1A–D, respectively), Tris-EDTA buffer (pH = 9.0) significantly improved staining signals of p-Tau (Figure 1E) and α-Syn (Figure 1F). In contrast, no significant improvement was found for Aβ staining (Figure 1G), while p-TDP43 staining even exhibited increased non-specific nuclear staining and even darker background (Figure 1H).

Further optimization of α-Syn IHC staining protocol

By comparing the three antigen retrieval conditions for α-Syn staining: i) boiling in Tris-EDTA buffer (pH = 9.0) alone for 15 min (Figure 2A); ii) incubate with 80% formic acid alone for 15 min at RT (Figure 2B); and iii) sequential treatment with boiling Tris-EDTA buffer (pH = 9.0, 15 min) followed by 80% formic acid (15 min, RT), the sequential treatment yielded the strongest α-Syn signals (Figure 2C).

Additionally, with the sequential treatment, extending the boiling time in Tris-EDTA buffer (pH = 9.0) from 15 min to 20 min or 30 min (Figure 2D and E) resulted in loss of inclusion and thread staining (Figure 2C). With 15 min of treatment in boiling Tris-EDTA buffer (pH = 9.0), shortening the incubation time in 80% formic acid from 15 min to 10 min (Figure 2F) showed no significant difference for the staining result (Figure 2C). Therefore, Tris-EDTA buffer (pH = 9.0, 15 min) followed by 80% formic acid (10 min, RT) which had the shorter antigen retrieval time and the ideal staining result was chosen as the optimal protocol.

Further optimization of Aβ IHC staining protocol

For Aβ staining with BAM-10 antibody, sequential treatment of boiling in citric acid buffer (pH = 6.0, 15 min) and then incubation in 80% formic acid (10 min, RT) slightly enhanced the Aβ plaque signals (Figure 3B) compared with treatment using boiling citric acid buffer alone (pH = 6.0, 15 min, Figure 3A), although it was noted that neither condition generated sufficiently strong signals. We further applied this sequential treatment using 6E10 and 6F3D antibodies. Both 6E10 (Figure 3C) and 6F3D (Figure 3D) showed stronger plaque signals with clearer backgrounds than BAM-10 (Figure 3B). Therefore, antibody 6F3D with boiling citric acid buffer (pH = 6.0, 15 min) followed by 80% formic acid incubation (10 min, RT) was chosen for the optimal protocol due to its clearer background than 6E10.

Further optimization of p-TDP43 IHC staining protocol

Following antigen retrieval with boiling citric acid buffer (pH = 6.0, 15 min), a serial dilution of the p-TDP43 primary antibody (1:1000, 1:2000, 1:3000, and 1:4000) was applied in the frontal cortex (Figure 4A–D). Among these dilutions, 1:3000 was found to show the optimal staining results, characterized by clear signals and clear background (Figure 4C). This dilution was subsequently validated across multiple brain regions in another CBB-ZJU case, including the amygdala (Figure 4E), frontal cortex (Figure 4F), hippocampal dentate gyrus (Figure 4G), and CA1 (Figure 4H).

Validation of optimized IHC staining protocols

The optimized CBB-ZJU IHC staining protocols for p-Tau, α-Syn, Aβ, and p-TDP43 are summarized in Table 3.

To validate these protocols, hippocampal (for Aβ and p-Tau staining) and amygdala (for p-TDP43 and α-Syn staining) tissue sections obtained from NBB and CBB-ZJU were used for staining. Sections from NBB were stained using the optimized CBB-ZJU IHC staining protocols for p-Tau, α-Syn, Aβ, and p-TDP43 (Figure 5A–D, respectively). The results demonstrated high consistency in staining intensity, specificity, or distribution with those stained in NBB (Figure 5E–H, respectively). In addition, sections from CBB-ZJU were sent to NBB for staining using NBB IHC staining protocols for p-Tau, α-Syn, Aβ, and p-TDP43 (Figure 5M–P, respectively), which showed no significant differences compared with those stained at CBB-ZJU using the optimized protocols (Figure 5I–L, respectively).

Discussion

The present study demonstrates that the IHC staining with the original protocols of CBB-ZJU were significantly improved by a few alterations that facilitated detection of the four general tested neuropathological proteins, i.e., p-Tau, α-Syn, Aβ, and p-TDP43. It should be noted that our validation primarily utilized representative cases demonstrating common pathologies (e.g., Aβ plaques and neurofibrillary tangles) on formalin-fixed, paraffin-embedded archival post-mortem human brain tissues. Therefore, cases with the other pathological morphologies, such as cerebral amyloid angiopathy (CAA) and ageing-related Tau astrogliopathy (ARTAG), were not included in this study. Nevertheless, in routine pathological diagnostic work at CBB-ZJU, our optimized IHC staining protocols are capable of reliably identifying their related pathological changes, namely, amyloid deposition within cerebral vessels in CAA and Tau positive astrocytes for aging-related Tau astrogliopathy (ARTAG). The reliability and repeatability of the optimized IHC staining protocols were verified through consistent results, as determined by comparison with reference samples from the NBB.

Fixation of brain tissue in 10% formalin solution (35%–40% formaldehyde solution) leads to protein cross-linking, since formaldehyde causes covalent bonds among proteins and protein-nucleic acid cross-links in brain tissue[21]. Such cross-linking may restrict the entry of antibodies into tissue[21]. In addition, it alters the epitope three-dimensional structure of antigens, reducing the accessibility of antigenic determinant cluster to the antibody[22,23]. Therefore, the antigen retrieval step is crucial for IHC staining protocols for brain baking, which usually include enzymatic digestion, heating (using autoclave, microwave, oven, etc.), or formic acid pretreatment[22].

The major mechanism of heat-induced antigen retrieval is to break the cross-linking among proteins caused by formalin, and the pH value of the retrieval solution is a key factor for restoring the antigenic determinants for related antibodies[24]. Consequently, the efficiency of antigen retrieval is highly dependent on the pH value of the retrieval solution[25]. Previous studies have systematically characterized the effects of pH of antigen retrieval solutions[25], as summarized below: Type A, or “the stable type”, meaning for the IHC staining results keeping almost the same with any pH (pH = 1.0–10.0) of antigen retrieval solutions; For Type B, the IHC staining intensity decreases at pH 3.0–6.0 and increases at pH values above 6.0, while for Type C, the intensity increases steadily across the pH range of 1.0–10.0[25]. A study performed on different mouse tissues, including the uterine, ovarian, epididymal, liver, small intestine, kidney, and pancreas tissues, found that for most antibodies, the effect of pH value of antigen retrieval solution seen was type B, while sometimes it was type C[26]. In addition, it was found that the pH-dependent heat-induced antigenic retrieval may hold for tissues from different species, including human and mouse[26]. A previous study has demonstrated that the antigenicity of most antibodies could be retrieved when heated in acidic (pH = 3.0) or alkaline (pH = 9.0 and pH = 10.5) buffers, whereas pH = 7.5 did not yield satisfactory antigen retrieval under the same experimental parameters[26]. Therefore, in the present study, we employed two pH conditions (pH = 6.0 and pH = 9.0) for antigen retrieval.

The time for antigen retrieval may also be important. However, we observed that the staining intensity of α-Syn showed no significant difference among incubations in boiling Tris-EDTA buffer (pH 9.0) for 15, 20, or 30 min, nor between incubations in 80% formic acid for 10 and 15 min. The optimal boiling time for α-Syn antigen retrieval was 15 min, which is in consistent with the reports showing that heat exposure for 10–20 min provide a good staining of human brain tissue that has been fixed in formaldehyde solution for a few hours, several days, or even several months[27].

A previous study found that formic acid may facilitate antigenic retrieval for the visualization of extracellular Aβ deposits in human brain tissues with long-fixation time of 14 years[22]. Our present study confirmed that formic acid improved the intensity of the immunoactivity of Aβ. In addition, we showed that different clones of Aβ antibodies show different IHC staining results[8]. Using our optimized Aβ IHC staining protocol of using boiling antigen retrieval solution citric acid buffer (pH = 6.0) for 15 min followed by 80% formic acid at RT for 10 min, the clone of 6F3D Aβ-antibody showed the best staining result with clear signals of Aβ plaque deposition and/or vascular Aβ deposition with a clean background.

For IHC staining of α-Syn, we combined boiling Tris-EDTA antigen retrieval solution (pH = 9.0) for 15 min, followed by 80% formic acid for 10 mins. This is in agreement with previous findings that α-Syn immunoreactivity can be obtained in human brain treated with formic acid[28,29] or with boiling Tris-EDTA buffer (pH = 9.0)[22,29].

There are multiple potential phosphorylation sites within human TDP-43, including 41 Serine, 15 Threonine, and 7 Tyrosine residues. Notably, abnormal phosphorylation mainly takes place at serine residues in the carboxyl (C)-terminal region of TDP43, including S379, S403/404, S409, S410, and S409/410, while phosphorylation at serine 409/410 (pS409/410) is a pathological hallmark of TDP-43 proteinopathies[30–33]. The antibody of pTDP-43 (pS409/410) should be used for detecting disease-specific distribution patterns of pTDP-43 pathology for diagnosis purposes. We observed that brain tissue sections in boiling citric acid buffer (pH = 6.0) for 15 min may result in an optimal staining result that helps the neuropathological diagnosis for frontotemporal lobar degeneration and amyotrophic lateral sclerosis[25,31].

Concern has been raised regarding whether unilateral sampling for neuropathological diagnosis yields consistent results with those from the contralateral hemisphere, while to date, no published literature has investigated this question. Since its establishment, the CBB-ZJU has strictly adhered to the standard operating protocols of the NBB. To address these points, we are currently developing a protocol for alternating fixation of the left and right hemispheres and plan to initiate a systematic investigation into lateralization in the neuropathological diagnosis.

The consistent staining results between CBB-ZJU and NBB showed that CBB-ZJU has basically the standardization and repeatability of the staining process, reliability of sample quality, compatibility of antibodies and reagents. In addition, it confirms the equal level of quality control between CBB-ZJU and international advanced brain banks such as NBB.

In summary, as a key tool for neuropathological diagnosis in human brain banking, the optimized and validated IHC staining protocols for p-Tau, α-Syn, Aβ, and p-TDP43 resulted from the present study may help to establish a globally consistent neuropathological diagnostic system among national and international human brain banks.

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