Materials and Reagents

Pipettes (M&S Instruments, catalog numbers: F144059M, F144058, and F144055M)
Pipette tips (Violamo, catalog numbers: V-1000, V-200, and V-10)
Eppendorf centrifuge tubes, 1.5 mL
6 cm dishes (AS ONE, catalog number: 2-8590-02)
Phosphate buffer saline (PBS) (FUJIFILM, catalog number: 166-23555)
Dimethyl sulfoxide (FUJIFILM, catalog number: 041-29351)
Normal saline (Otsuka Pharmaceutical Factory, catalog number: 035081517)
ProteoGREEN^TM-gGlu (GORYO Chemical, catalog number: GC801). Dissolve in dimethyl sulfoxide at 1 mM and store at -20 °C before use (unnecessary to filter)

Equipment

Stereomicroscope (BioTools, catalog number: BS-3048BT)
Fluorescent unit (BioTools, catalog number: BT-ExSM)
Band pass filter (FUJIFILM, catalog number: BPB-45). This filter should be set at the light source of the fluorescent unit
Sharp cut filter (FUJIFILM, catalog number: SC-52). This filter should be set at the lens side of the fluorescent unit (Figure 1)

Figure 1. Setup of the fluorescent unit for the imaging. Band pass filter and sharp cut filter should be set as illustrated.

Procedure

Preparation of the fluorescent probe
1. Thaw ProteoGREEN^TM-gGlu stock solution on ice (protect from light).
2. Dilute ProteoGREEN^TM-gGlu stock solution into PBS to prepare fluorescent solution (concentration of 50 μM).
Handling of renal biopsy specimen
1. Perform renal biopsy (Donovan et al., 1991).
2. Briefly rinse biopsy specimen with 10 mL of normal saline in 6 cm dish. Protect the specimen from drying.
3. Apply 100 μL of the fluorescent solution to the biopsy specimen. Incubate for 3 min under dark conditions at room temperature.
4. Illuminate and observe the specimen. Excitation wavelength of 450 nm passes through the band pass filter; the fluorescent solution is excited and emits fluorescence. Sharp cut filter eliminates redundant wavelengths except for fluorescence.
5. Capture images of the renal biopsy specimen at 2.0× magnification ratio. The corticomedullary junction of the specimen can be easily identified (Figure 2).
Figure 2. Fluorescent image of the renal biopsy specimen incubated with gGlu-HMRG. Cortex emits stronger fluorescence than the medulla. Scale bar = 1 mm.

Notes

Prepare fluorescent solution each time just before use.
Fluorescent solution should be applied to the specimen before fixation.
Renal carcinoma deriving from the renal proximal tubular epithelial cells might influence the fluorescence.

Acknowledgments

This protocol is based on “A novel method for assessing the renal biopsy specimens using an activatable fluorescent probe,” published in Scientific Reports (Iyama et al., 2020).

Competing interests

The authors declare that they have no financial or non-financial conflicts of interest.

Ethics

This study was conducted in accordance with the Declaration of Helsinki and approved by the ethical committee of Tottori University Hospital (approval number: 18A135). Informed consent was obtained from all subjects.

References

Iyama, T., Takata, T., Yamada, K., Mae, Y., Taniguchi, S., Ida, A., Ogawa, M., Yamamoto, M., Hamada, S., Fukuda, S., et al. (2020). A novel method for assessing the renal biopsy specimens using an activatable fluorescent probe. Sci Rep 10(1): 12094.
Mitsunaga, M., Kosaka, N., Choyke, P. L., Young, M. R., Dextras, C. R., Saud, S. M., Colburn, N. H., Sakabe, M., Nagano, T., Asanuma, D., et al. (2013). Fluorescence endoscopic detection of murine colitis-associated colon cancer by topically applied enzymatically rapid-activatable probe. Gut 62(8): 1179-1186.
Urano, Y., Sakabe, M., Kosaka, N., Ogawa, M., Mitsunaga, M., Asanuma, D., Kamiya, M., Young, M. R., Nagano, T., Choyke, P. L., et al. (2011). Rapid cancer detection by topically spraying a gamma-glutamyltranspeptidase-activated fluorescent probe. Sci Transl Med 3(110): 110ra119.
Donovan, K. L., Thomas, D. M., Wheeler, D. C., Macdougall, I. C. and Williams, J. D. (1991). Experience with a new method for percutaneous renal biopsy. Nephrol Dial Transplant 6(10): 731-733.