Expansion Pathology: A Method for High-resolution Optical Imaging of Clinical Tissue Samples

Published: April 30, 2023

Abstract

Source: Klimas, A. et al. Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion. J. Vis. Exp. (2019)

In this video, we describe a method called expansion pathology, a variant of expansion microscopy, wherein the clinical tissue specimens are chemically expanded using a polymer network for microscopic examination of nanoscale structures.

Protocol

1. In Situ Polymerization of Specimens Incubate the specimen in an anchoring solution. Prepare the anchoring solution (typically 250 µL is enough to cover the tissue section) by diluting the AcX stock solution in 1x PBS to a concentration of 0.03 mg/mL for samples fixed with non-aldehyde fixatives or 0.1 mg/mL for samples fixed with aldehyde fixatives, which have fewer free amines available to react with AcX. Place the slide in a 100 mm Petri dish and pipette the a…

Representative Results

Figure 1: Gelation chamber for pathology samples. (A) Two spacers, such as two pieces of #1.5 cover glass cut with a diamond knife, are placed on either side of the tissue after incubating the sample in gelling solution at 4 °C. The spacers should be thicker than the tissue slices, to prevent compression of the sample. (B) A lid, such a…

Divulgaciones

The authors have nothing to disclose.

Materials

6-well glass-bottom plate (#1.5 coverglass)  Cellvis  P06-1.5H-N
Acetone  Fischer Scientifc  A18-500
Acrylamide  Sigma Aldrich  A8887
Acryloyl-X SE, (AcX)  Invitrogen  A20770
Agarose  Fischer Scientifc  BP160-100
Ammonium persulfate (APS)  Sigma Aldrich  A3678  Initiatior
DAPI (1 mg/mL)  Thermo Scientific  62248  Nuclear stain
Diamond knife No. 88 CM  General Tools  31116
Ethanol  Pharmco  111000200
Ethylenediaminetetraacetic acid (EDTA) 0.5 M  VWR  BDH7830-1
FFPE Kidney Sample  USBiomax  HuFPT072
Forceps
Micro cover Glass #1 (24 mm x 60 mm)  VWR  48393 106
Micro cover Glass #1.5 (24 mm x 60 mm)  VWR  48393 251
N,N,N′,N′-Tetramethylethylenediamine (TEMED)  Sigma Aldrich  T9281  Accelerator
N,N′-Methylenebisacrylamide  Sigma Aldrich  M7279
Nunclon 6-Well Cell Culture Dish  Thermo Fisher  140675
Nunc 15 mL Conical  Thermo Fisher  339651
Nunc 50 mL Conical  Thermo Fisher  339653
Orbital Shaker
Paint brush
pH Meter
Phosphate Buffered Saline (PBS) 10x Solution  Fischer Scientifc  BP399-1
Plastic Petri Dish (100 mm)  Fischer Scientifc  FB0875713
Proteinase K (Molecular Biology Grade)  Thermo Scientific  EO0491
Razor blade  Fischer Scientifc  12640
Safelock Microcentrifuge Tubes 1.5 mL  Thermo Fisher  3457
Safelock Microcentrifuge Tubes 2.0 mL  Thermo Fisher  3459
Sodium acrylate  Sigma Aldrich  408220
Sodium chloride  Sigma Aldrich  S6191
Sodium citrate tribasic dihydrate  Sigma Aldrich  C8532-1KG
Tris Base  Fischer Scientifc  BP152-1
Triton X-100  Sigma Aldrich  T8787
Xylene  Sigma Aldrich  214736

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Expansion Pathology: A Method for High-resolution Optical Imaging of Clinical Tissue Samples. J. Vis. Exp. (Pending Publication), e20576, doi: (2023).

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