HQ-O

ABSTRACT:
Background: Various methodologies have been employed for the localization of amyloid plaques in numerous studies on Alzheimer’s disease. The majority of these stains are thought to label the plaques by virtue of their affinity for aggregated Aβ. However, plaques are known to contain numerous other components, including multivalent metals such as zinc. A novel fluorescent zinc chelator, HQ-O, was investigated to determine its mechanism of binding and to optimize a stain for the high contrast and resolution histological localization of amyloid plaques. The histology involves incubating tissue sections in a dilute aqueous solution of HQ-O. Its compatibility with a variety of other fluorescent methodologies is described. All amyloid plaques are stained in fine detail and appear bright green under blue light excitation. The staining of parenchymal plaques correlates closely with that seen following staining with antibodies to Aβ; however, the HQ-O sometimes also label additional globular structures within blood vessels. The tracer is also capable of labeling intravascular leucocytes, when present, presumably due to their high zinc content.

Objective: To investigate whether it is possible to localize the presence of zinc in parenchymal and vascular amyloid plaques in afflicted brains. To accomplish this, a novel fluorescent zinc chelator, HQ-O, was investigated to determine its mechanism of binding and to optimize a stain for the high contrast and resolution histological localization of amyloid plaques.

Method: A novel zinc chelator, HQ-O, was developed for localizing zinc containing amyloid plaques. The histology involves incubating tissue sections in a dilute aqueous solution of HQ-O. Its compatibility with a variety of other fluorescent methodologies is described.

Results: All amyloid plaques are stained in fine detail and appear bright green under blue light excitation. The staining of parenchymal plaques correlates closely with that seen following staining with antibodies to Aβ; however, the HQ-O sometimes also label additional globular structures within blood vessels. In situ mechanistic studies revealed that fluorescent plaque-like structures are only observed with HQ-O when synthetic Aβx-42 is aggregated in the presence of zinc.

Conclusions: Zinc is intimately bound to all amyloid plaques, which was demonstrated by its histological localization using a novel fluorescent zinc chelator, HQ-O. Additionally, the tracer is also capable of labeling intravascular leucocytes due to their high zinc content.

HQ-O HISTOLOGICAL STAINING PROTOCOL:
HQ-O Staining: Frozen sections are mounted onto gelatin-coated slides and allowed to dry on a slide warmer for at least 20 min. Typically, the slides are first immersed in 100% ethanol for 5 min followed by 3 min in 70% ethanol and 3 min in distilled water. Should exposure to solvents be counter-indicated, this step may be omitted, and the sections may simply be rehydrated in distilled water for 3 min. The slides are then transferred to the HQ-O staining solution which is prepared by adding 33 mg of HQ-O (Histo-Chem, Jefferson AR) to 100 ml of distilled water vehicle. The optimal staining time is temperature dependent, therefore overnight (16-24 hrs.) incubation is required when staining fresh tissue sections at room temperature, while only 3 hrs. incubation time is required at 60°C. The staining solution should be used within 24 hrs. of preparation. Paraffin-embedded sections are first deparaffinized in xylene and then rehydrated with a graded series of alcohols and finally in distilled water. The hydrated slides are then transferred to the same HQ-O staining solution as described above. At room temperature, the paraffin-processed tissue will require around 3 hrs. to fully stain, while tissue sections incubated at 60°C will require only about 45 min to stain. Following staining, all slides are rinsed for 3 min through 2 changes of distilled water and then allowed to air dry on a slide warmer. Slides are cleared by brief (1-2 min) immersion in xylene and then coverslipped with DPX (Sigma-Aldrich, St. Louis MO), a non-polar and non-fluorescent mounting media. Slides are visualized under blue light excitation.

REFERENCE:
Schmued, LC, Raymick, J, Sarkar, S; High contrast and resolution labeling of amyloid plaques in tissue sections from APP-PS1 mice and humans with Alzheimer’s Disease with the zinc chelator HQ-O: Practical and theoretical considerations, Curr. Alzheimer Res. 2019.

View Sample Photomicrographs