Postmortem human brains are important research resources to study the mechanisms underlying cerebrovascular features in various neurodegenerative disorders. Immunohistochemical and histochemical staining have been used to examine human brains fixed in neutral-buffered formalin (NBF) for months, years, or decades. Previously, we found that prolonged NBF fixation resulted in differential effects on immunohistochemistry and histochemistry staining on postmortem brains. Here, we further examined the effects of prolonged fixation (1, 5, 10, 15, and 20 years) on stains of known biomarkers of cerebrovascular diseases in the human prefrontal cortex. We included microvasculature markers of the blood vessel wall (anti-collagen-IV and claudin-5), a type III intermediate filament marker (anti-vimentin), an activated microglia marker (anti-CD68), a biomarker for proteolipid protein (anti-PLP) of oligodendrocytes and a marker for iron accumulation (anti-ferritin). We also included Masson’s trichrome stain (MTS) and Bielschowsky silver stain (BSS). We found that staining intensities of ferritin, vimentin, collagen-IV, and BSS decreased with prolonged fixation. No significant differences were observed in the staining intensity of other markers. We therefore recommend performing IHC and HC staining for human brains with the same fixation times to offset any impact on downstream neuropathological analyses, as well as adding the fixation duration as a covariate in the analysis.
Cortes-CanteliMIadecolaC. Alzheimer’s disease and vascular aging: JACC focus seminar. J Am Coll Cardiol2020; 75(8): 942–951.
2.
SabbatinelliJRaminiDGiulianiA, et al. Connecting vascular aging and frailty in Alzheimer’s disease. Mech Ageing Dev2021; 195: 111444.
3.
ArvanitakisZCapuanoALeurgansSE, et al. Relation of cerebral vessel disease to Alzheimer’s disease dementia and cognitive function in elderly people: a cross-sectional study. Lancet Neurol2016; 15(9): 934–943.
4.
DadarMMahmoudSZhernovaiaM, et al. White matter hyperintensity distribution differences in aging and neurodegenerative disease cohorts. Neuroimage Clin2022; 36: 103204.
5.
WardlawJMValdés HernándezMCMuñoz-ManiegaS. What are white matter hyperintensities made of? Relevance to vascular cognitive impairment. J Am Heart Assoc2015; 4(6): 001140.
6.
CardosoJRPereiraLMIversenMD, et al. What is gold standard and what is ground truth?. Dental Press J Orthod2014; 19(5): 27–30.
7.
Durand-MartelPTremblayDBrodeurC, et al. Autopsy as gold standard in FDG-PET studies in dementia. Can J Neurol Sci2010; 37(3): 336–342.
8.
HumphreysCAJansenMAManiegaSM, et al. A protocol for precise comparisons of small vessel disease lesions between ex vivo magnetic resonance imaging and histopathology. Int J Stroke2019; 14(3): 310–320.
9.
HoffmannABrednoJWendlandMF, et al. Validation of in vivo magnetic resonance imaging blood–brain barrier permeability measurements by comparison with gold standard histology. Stroke2011; 42(7): 2054–2060.
10.
KovacsGGBudkaH. Current concepts of neuropathological diagnostics in practice: neurodegenerative diseases. Clin Neuropathol2010; 29(5): 271–288.
11.
SukswaiNKhouryJD. Immunohistochemistry innovations for diagnosis and tissue-based biomarker detection. Curr Hematol Malig Rep2019; 14(5): 368–375.
12.
AdickesEDFolkerthRDSimsKL. Use of perfusion fixation for improved neuropathologic examination. Arch Pathol Lab Med1997; 121(11): 1199–1206.
13.
StumptnerCPabstDLoibnerM, et al. The impact of crosslinking and non-crosslinking fixatives on antigen retrieval and immunohistochemistry. N Biotechnol2019; 52: 69–83.
14.
FrigonEMDadarMBoireD, et al. Antigenicity is preserved with fixative solutions used in human gross anatomy: a mice brain immunohistochemistry study. Front Neuroanat2022; 16: 45–63.
15.
FrigonEMGérin-LajoieADadarM, et al. Comparison of histological procedures and antigenicity of human post-mortem brains fixed with solutions used in gross anatomy laboratories. Front Neuroanat2024; 18: 1372953.
16.
MaWFrigonEMMaranzanoJ. Differential effects of prolonged post-fixation on immunohistochemical and histochemical staining for postmortem human brains. Front Neuroanat2024; 18: 85–96.
17.
HadeACPhilipsMAPrometL, et al. A cost-effective and efficient ex vivo, ex situ human whole brain perfusion protocol for immunohistochemistry. J Neurosci Methods2024; 404: 110059.
18.
BeachTGAdlerCHSueL, et al. Arizona Study of Aging and Neurodegenerative Disorders and Brain and Body Donation Program. Neuropathology2015; 35(4): 354–389.
19.
VonsattelJPdel Pilar AmayaMCortesEP, et al. Twenty-first century brain banking: practical prerequisites and lessons from the past: the experience of New York Brain Bank, Taub Institute, Columbia University. Cell Tissue Bank2008; 9(3): 247–258.
20.
McFaddenWCWalshHRichterF, et al. Perfusion fixation in brain banking: a systematic review. Acta Neuropathol Commun2019; 7(1): 146.
21.
WeisbeckerV. Distortion in formalin-fixed brains: using geometric morphometrics to quantify the worst-case scenario in mice. Brain Struct Funct2011; 217: 677–685.
22.
HelanderKG. Kinetic studies of formaldehyde binding in tissue. Biotech Histochem1994; 69(3): 177–179.
23.
ThavarajahRMudimbaimannarVKElizabethJ, et al. Chemical and physical basics of routine formaldehyde fixation. J Oral Maxillofac Pathol2012; 16(3): 400–405.
24.
ArnoldMMSrivastavaSFredenburghJ, et al. Effects of fixation and tissue processing on immunohistochemical demonstration of specific antigens. Biotech Histochem1996; 71(5): 224–230.
25.
JiaoYSunZLeeT, et al. A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections. J Neurosci Methods1999; 93(2): 149–162.
26.
Ramos-VaraJABeissenherzME. Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed paraffin-embedded tissues: experience with 63 markers. J Vet Diagn Invest2000; 12(4): 307–311.
27.
ShiSRCoteRJTaylorCR. Antigen retrieval immunohistochemistry used for routinely processed celloidin-embedded human temporal bone sections: standardization and development. Auris Nasus Larynx1998; 25(4): 425–443.
28.
ShiSRShiYTaylorCR. Antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades. J Histochem Cytochem2011; 59(1): 13–32.
29.
AlrafiahAAlshaliR. The effect of prolonged formalin fixation on the staining characteristics of archival human brain tissue. Folia Morphol2019; 78(2): 230–236.
30.
LyckLDalmauIChemnitzJ, et al. Immunohistochemical markers for quantitative studies of neurons and glia in human neocortex. J Histochem Cytochem2008; 56(3): 201–221.
31.
WebsterJDMillerMADuSoldD, et al. Effects of prolonged formalin fixation on diagnostic immunohistochemistry in domestic animals. J Histochem Cytochem2009; 57(8): 753–761.
32.
WuXDengCSuY, et al. The effect of prolonged formalin fixation on the expression of proteins in human brain tissues. Acta Histochem2022; 124(4): 151879.
33.
ToviMEricssonA. Measurements of T1 and T2 over time in formalin-fixed human whole-brain specimens. Acta Radiol1992; 33(5): 400–404.
34.
DadarMSanchesLFouquetJP, et al. The Douglas–Bell Canada Brain Bank post-mortem brain imaging protocol. Aperture Neuro2024; 4: 45–68.
35.
HymanBTPhelpsCHBeachTG, et al. National Institute on Aging–Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease. Alzheimers Dement2012; 8(1): 1–13.
36.
ShuSYJuGFanLZ. The glucose oxidase–DAB–nickel method in peroxidase histochemistry of the nervous system. Neurosci Lett1988; 85(2): 169–171.
37.
BenjaminiYHochbergY. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B Methodol1995; 57(1): 289–300.
38.
KiernanJA. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Microsc Today2000; 8(1): 8–13.
39.
Gallardo-CaballeroMRodriguez-MorenoCBAlvarez-MendezL, et al. Prolonged fixation and post-mortem delay impede the study of adult neurogenesis in mice. Commun Biol2023; 6(1): 978.
40.
LundströmYLundströmPPoponaSN, et al. Detection of changes in immunohistochemical stains caused by postmortem delay and fixation time. Appl Immunohistochem Mol Morphol2019; 27(3): 238–245.
41.
HoffmanEAFreyBLSmithLM, et al. Formaldehyde crosslinking: a tool for the study of chromatin complexes. J Biol Chem2015; 290(44): 26404–26411.
42.
HoppertonKEMohammadDTrépanierMO, et al. Markers of microglia in post-mortem brain samples from patients with Alzheimer’s disease: a systematic review. Mol Psychiatry2018; 23(2): 177–198.
43.
ZigmondREEchevarriaFD. Macrophage biology in the peripheral nervous system after injury. Prog Neurobiol2019; 173: 102–121.
44.
FarsoMMénardCColby-MilleyJ, et al. The immune marker CD68 correlates with cognitive impairment in normally aged rats. Neurobiol Aging2013; 34(8): 1971–1976.
45.
MinettTClasseyJMatthewsFE, et al. Microglial immunophenotype in dementia with Alzheimer’s pathology. J Neuroinflammation2016; 13(1): 135.
46.
van WeteringJGeutHBolJJ, et al. Neuroinflammation is associated with Alzheimer’s disease co-pathology in dementia with Lewy bodies. Acta Neuropathol Commun2024; 12(1): 73.
47.
MakkinejadNZanon ZotinMCvan den BrinkH, et al. Neuropathological correlates of white matter hyperintensities in cerebral amyloid angiopathy. J Am Heart Assoc2024; 13(22): e035744.
48.
ZotovaEHolmesCJohnstonD, et al. Microglial alterations in human Alzheimer’s disease following Aβ42 immunization. Neuropathol Appl Neurobiol2011; 37(5): 513–524.
49.
GreerJMPenderMP. Myelin proteolipid protein: an effective autoantigen and target of autoimmunity in multiple sclerosis. J Autoimmun2008; 31(3): 281–287.
50.
GreerJMTrifilieffEPenderMP. Correlation between anti-myelin proteolipid protein (PLP) antibodies and disease severity in multiple sclerosis patients with PLP response–permissive HLA types. Front Immunol2020; 11: 1891.
51.
KnappPE. Proteolipid protein: is it more than just a structural component of myelin?. Dev Neurosci1996; 18(4): 297–308.
52.
SimonsMKramerEMMacchiP, et al. Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes: implications for Pelizaeus–Merzbacher disease. J Cell Biol2002; 157(2): 327–336.
53.
RoherAEWeissNKokjohnTA, et al. Increased A beta peptides and reduced cholesterol and myelin proteins characterize white matter degeneration in Alzheimer’s disease. Biochemistry2002; 41(37): 11080–11090.
54.
NasrabadySERizviBGoldmanJE, et al. White matter changes in Alzheimer’s disease: a focus on myelin and oligodendrocytes. Acta Neuropathol Commun2018; 6(1): 22.
55.
AndersenHHJohnsenKBMoosT. Iron deposits in the chronically inflamed central nervous system and contributes to neurodegeneration. Cell Mol Life Sci2014; 71(9): 1607–1622.
56.
SimmonsDACasaleMAlconB, et al. Ferritin accumulation in dystrophic microglia is an early event in the development of Huntington’s disease. Glia2007; 55(10): 1074–1084.
57.
JusticiaCRamos-CabrerPHoehnM. MRI detection of secondary damage after stroke: chronic iron accumulation in the thalamus of the rat brain. Stroke2008; 39(5): 1541–1547.
58.
GuidouxCHauwJJKleinIF, et al. Amyloid angiopathy in brain hemorrhage: a postmortem neuropathological–magnetic resonance imaging study. Cerebrovasc Dis2018; 45(3–4): 124–131.
59.
SchragMCroftonAZabelM, et al. Effect of cerebral amyloid angiopathy on brain iron, copper, and zinc in Alzheimer’s disease. J Alzheimers Dis2011; 24(1): 137–149.
60.
SharmaBBeaudinAECoxE, et al. Brain iron content in cerebral amyloid angiopathy using quantitative susceptibility mapping. Front Neurosci2023; 17: 1139988.
61.
ConnorJRMenziesSLSt MartinSM, et al. Cellular distribution of transferrin, ferritin, and iron in normal and aged human brains. J Neurosci Res1990; 27(4): 595–611.
62.
DaglasMAdlardPA. The involvement of iron in traumatic brain injury and neurodegenerative disease. Front Neurosci2018; 12: 981.
63.
LongHZhuWWeiL, et al. Iron homeostasis imbalance and ferroptosis in brain diseases. MedComm (2020)2023; 4(4): e298.
64.
van DuijnSNabuursRJvan DuinenSG, et al. Comparison of histological techniques to visualize iron in paraffin-embedded brain tissue of patients with Alzheimer’s disease. J Histochem Cytochem2013; 61(11): 785–792.
65.
AntharamVCollingwoodJFBullivantJP, et al. High field magnetic resonance microscopy of the human hippocampus in Alzheimer’s disease: quantitative imaging and correlation with iron. Neuroimage2012; 59(2): 1249–1260.
66.
De BarrosAArribaratGCombisJ, et al. Matching ex vivo MRI with iron histology: pearls and pitfalls. Front Neuroanat2019; 13: 68.
67.
NagSChenEYJohnsonR, et al. Ex vivo MRI facilitates localization of cerebral microbleeds of different ages during neuropathology assessment. Free Neuropathol2021; 2: 35.
68.
SchragMMcAuleyGPomakianJ, et al. Correlation of hypointensities in susceptibility-weighted images to tissue histology in dementia patients with cerebral amyloid angiopathy: a postmortem MRI study. Acta Neuropathol2010; 119(3): 291–302.
69.
TatsumiSShinoharaMYamamotoT. Direct comparison of histology of microbleeds with postmortem MR images: a case report. Cerebrovasc Dis2008; 26(2): 142–146.
70.
LvJHuWYangZ, et al. Focusing on claudin-5: a promising candidate in the regulation of BBB to treat ischemic stroke. Prog Neurobiol2018; 161: 79–96.
71.
TachibanaKHirayamaRSatoN, et al. Association of plasma claudin-5 with age and Alzheimer disease. Int J Mol Sci2024; 25(3): 1419.
ChiuFCNortonWTFieldsKL. The cytoskeleton of primary astrocytes in culture contains actin, glial fibrillary acidic protein, and the fibroblast-type filament protein, vimentin. J Neurochem1981; 37(1): 147–155.
74.
SchnitzerJFrankeWWSchachnerM. Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system. J Cell Biol1981; 90(2): 435–447.
75.
KiddMEShumakerDKRidgeKM. The role of vimentin intermediate filaments in the progression of lung cancer. Am J Respir Cell Mol Biol2014; 50(1): 1–6.
76.
BayirESendemirA. Role of intermediate filaments in blood–brain barrier in health and disease. Cells, 2021; 10(6): 1400.
77.
ChenKZLiuSXLiYW, et al. Vimentin as a potential target for diverse nervous system diseases. Neural Regen Res2023; 18(5): 969–975.
78.
ZhangLWangJYanY, et al. Vimentin fragmentation and its role in amyloid-beta plaque deposition in Alzheimer’s disease. Int J Mol Sci2025; 26(7): 42–79.
79.
O’LearyLADavoliMABelliveauC, et al. Characterization of vimentin-immunoreactive astrocytes in the human brain. Front Neuroanat2020; 14: 31.
80.
MazzottiMCFaisPPalazzoC, et al. Determining the time of death by morphological and immunohistochemical evaluation of collagen fibers in postmortem gingival tissues. Leg Med2019; 39: 1–8.
81.
ChelloMMastrorobertoPZofreaS, et al. Analysis of collagen and elastin content in primary varicose veins. J Vasc Surg1994; 20(3): 490.
82.
KimHL. Arterial stiffness and hypertension. Clin Hypertens2023; 29(1): 31.
83.
WarehamLKBarattaRODel BuonoBJ, et al. Collagen in the central nervous system: contributions to neurodegeneration and promise as a therapeutic target. Mol Neurodegener2024; 19(1): 11.
84.
KumarAAYeoNWhittakerM, et al. Vascular collagen type-IV in hypertension and cerebral small vessel disease. Stroke2022; 53(12): 3696–3705.
85.
ÖzkanEÇetin-TasYSekerdagE, et al. Blood–brain barrier leakage and perivascular collagen accumulation precede microvessel rarefaction and memory impairment in a chronic hypertension animal model. Metab Brain Dis2021; 36(8): 2553–2566.
86.
DenverPD’AdamoHHuS, et al. A novel model of mixed vascular dementia incorporating hypertension in a rat model of Alzheimer’s disease. Front Physiol2019; 10: 1269.
87.
AllsopD. Introduction to Alzheimer’s disease. Methods Mol Med2000; 32: 1–21.
88.
ElobeidARantakömiSSoininenH, et al. Alzheimer’s disease-related plaques in nondemented subjects. Alzheimers Dement2014; 10(5): 522–529.
89.
FanSZhengYLiuX, et al. Curcumin-loaded PLGA–PEG nanoparticles conjugated with B6 peptide for potential use in Alzheimer’s disease. Drug Deliv2018; 25(1): 1091–1102.
90.
KlakotskaiaDAgcaCRichardsonRA, et al. Memory deficiency, cerebral amyloid angiopathy, and amyloid-β plaques in APP + PS1 double transgenic rat model of Alzheimer’s disease. PLoS One2018; 13(4): e0195469.
91.
LoveSNicollJA. Comparison of modified Bielschowsky silver impregnation and anti-ubiquitin immunostaining of cortical and nigral Lewy bodies. Neuropathol Appl Neurobiol1992; 18(6): 585–592.
92.
StahnischFW. Max Bielschowsky (1869–1940). J Neurol2015; 262(3): 792–794.
93.
SwitzerRCIII. Application of silver degeneration stains for neurotoxicity testing. Toxicol Pathol2000; 28(1): 70–83.
94.
UchiharaT. Silver diagnosis in neuropathology: principles, practice and revised interpretation. Acta Neuropathol2007; 113(5): 483–499.
95.
WisniewskiHMWenGYKimKS. Comparison of four staining methods on the detection of neuritic plaques. Acta Neuropathol1989; 78(1): 22–27.
96.
YamamotoTHiranoA. A comparative study of modified Bielschowsky, Bodian and thioflavin S stains on Alzheimer’s neurofibrillary tangles. Neuropathol Appl Neurobiol1986; 12(1): 3–9.
OnodaAHagiwaraSKubotaN, et al. A novel staining method for detection of brain perivascular injuries induced by nanoparticle: periodic acid-Schiff and immunohistochemical double-staining. Front Toxicol2022; 4: 825984.
99.
BearerEL. Exploring vascular contributions to cognitive impairment: small-vessel disease of white matter and microplastics/nanoplastics. Am J Pathol2025; 195(11): 2059–2074.
100.
FazekasFKleinertROffenbacherH, et al. The morphologic correlate of incidental punctate white matter hyperintensities on MR images. AJNR Am J Neuroradiol1991; 12(5): 915–921.
101.
KeithJGaoFQNoorR, et al. Collagenosis of the deep medullary veins: an underrecognized pathologic correlate of white matter hyperintensities and periventricular infarction?. J Neuropathol Exp Neurol2017; 76(4): 299–312.
102.
Baker-NighAVahediSDavisEG, et al. Neuronal amyloid-β accumulation within cholinergic basal forebrain in ageing and Alzheimer’s disease. Brain2015; 138(Pt 6): 1722–1737.
103.
FrigerioIBoonBDCLinCP, et al. Amyloid-β, p-tau and reactive microglia are pathological correlates of MRI cortical atrophy in Alzheimer’s disease. Brain Commun2021; 3(4): fcab281.
104.
HampelHHardyJBlennowK, et al. The amyloid-β pathway in Alzheimer’s disease. Mol Psychiatry2021; 26(10): 5481–5503.
105.
MarshSEBlurton-JonesM. Examining the mechanisms that link β-amyloid and α-synuclein pathologies. Alzheimers Res Ther2012; 4(2): 11.
106.
PikkarainenMMartikainenPAlafuzoffI. The effect of prolonged fixation time on immunohistochemical staining of common neurodegenerative disease markers. J Neuropathol Exp Neurol2010; 69(1): 40–52.
107.
SunYIpPChakrabarttyA. Simple elimination of background fluorescence in formalin-fixed human brain tissue for immunofluorescence microscopy. J Vis Exp2017(127): E56188.
108.
OliveiraVCCarraraRCVSimoesDLC, et al. Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections. Histol Histopathol2010; 25(8): 1017–1024.
109.
WangSRenXWangJ, et al. Blocking autofluorescence in brain tissues affected by ischemic stroke, hemorrhagic stroke, or traumatic brain injury. Front Immunol2023; 14: 1168292.
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