Addressing the Challenges of Multi-Immunofluorescent Deep Tissue Imaging of Islets and Vasculature in the Rat Pancreas

Rapid Communication

J Mol Biol & Mol Imaging. 2015; 2(2): 1022.

Addressing the Challenges of Multi-Immunofluorescent Deep Tissue Imaging of Islets and Vasculature in the Rat Pancreas

Naidoo V, Lang DM and Van der Merwe EL*

Department of Human Biology, University of Cape Town, South Africa

*Corresponding author: Van der Merwe EL; Department of Human Biology, University of Cape Town, Faculty of Health Sciences, Private Bag, Observatory, 7935, South Africa

Received: November 15, 2015; Accepted: December 03, 2015; Published: December 07, 2015


Whilst whole-mount (WM) multi-immunofluorescence is useful for studying selected micro anatomical relationships in three dimensions, our studies on pancreatic tissues revealed that signals of different fluorophores for localizing distinct structures were not equally detectable at all optical planes. We thus sought to determine the underlying reasons for this observation. Multiple fluorophores covering the blue to far red emission spectra were used to detect islets (insulin - AMCA/Cy2/Cy3) and pancreatic vasculature (Lycopersicon esculentum lectin-FITC, Collagen IV/Cy5, α-SMA/Alexa Fluor®488 or α-SMA/ Alexa Fluor®647). Regardless of antibody concentration or mountant, insulin was only detectable up to ≈66μM with AMCA. Nuclei stained by Hoechst 33342 were detected deeper (≈130μM), suggesting that internal absorption was unlikely the main reason for the poor detectability of AMCA. By contrast, insulin/Cy2 was detectable much deeper in WMs mounted in methyl salicylate (maximum ≈300μM), but many of these islets showed stronger staining on the superficial plane than seen internally. Insulin/Cy2 islets were barely detectable in glycerol (GL). Collagen IV/Cy5 detected vasculature at deeper planes in salicylate, more so than some of the aforementioned antibodies/fluorophores for vasculature detection, but the grainy resolution meant it was unsuitable for future experiments. By contrast, insulin/Cy3and lectin-FITC were readily detectable in both mountants but more superior image quality was shown in glycerol-mounted tissues at deeper optical planes (maximum ≈213μM). We thus conclude that insulin/Cy3 and lectin-FITC provided the most promising antibody combination for detectability and resolution of insulin in β-cells in islets and vasculature, respectively, down to ≈200μM in the WMs.

Keywords: 3-D imaging; Multi-immunofluorescence; Deep tissue imaging; Islets; Fluorophores; Optical clearing


3-D: Three-dimensional; α-SMA: Alpha-Smooth Muscle Actin; AMCA: Aminomethylcoumarin Acetate; β-cells: Beta cells; BSA: Bovine Serum Albumin; CLSM: Confocal Laser Scanning Microscope; Cy: Cyanine; DABCO: 1,4-Diazabicyclo[2.2.2]octane; DPSS: Diode-pumped Solid-State; ECRA: Ethics Committee for Research on Animals; GL: Glycerol; GP: Guinea Pig; HeNe: Helium Neon; hr: hours; JIR: Jackson Immuno Research; Lectin-FITC: Lycopersicon esculentum Lectin-FITC; min: minutes; nm: Nanometer; NA: Numerical Aperture; PBS: Phosphate Buffered Saline; PFA: Paraformaldehyde; SAL: Methyl Salicylate; SA MRC: South African Medical Research Council; TX-100: Triton X-100; WEM: Wide-field Epifluorescence Microscopy; WM: Whole-Mount; viz.: Namely; μM: Micron


Multiple immunofluorescent staining and 3-D imaging of pancreatic whole-mounts (WMs) has gained popularity for better understanding of the micro-anatomical and cellular relationships relating to the islet neurovascular complexes [1-9]. Despite the promise of this approach, our studies on multi-immunostained rat pancreatic WMs revealed that not all immunostained structures of interest are equally detectable at optical planes deeper in the tissue. For our studies, we needed to locate islets in WMs under wide-field epifluorescence microscopy (WEM) before progressing to confocal imaging. Because we expected insulin content to be abundant in β-cells in the normal pancreas, we chose to detect islets using AMCA-conjugated secondary antibodies. The capillary network and other antigens of interest would thus be detected with Lectin- FITC and Cy3/Cy5/Alexa Fluor488/Alexa Fluor647-conjugated secondary antibodies respectively. Possible reasons that could account for the differential detection of fluorophores on structures known to be present at the deeper planes in the tissue are: insufficient permeabilization and antibody concentrations [10,11]; insufficient antibody incubation periods; weak-emitting fluorophores and antibodies that degrade over time [12]; internal absorption of shorter emission wavelengths due to refractive properties of the tissue (Table 1) [13]; the effect of mounting media (polar vs. non-polar), pH and optical clarity of the tissue on signal brightness [13,14]; and the size of antibody-fluorophore conjugates which may lead to steric hindrance or differences in penetration rate into the tissue. This study attempts to address several of the above challenges, focussing on the ability to detect pancreatic islets by WEM, and examine islet vasculature (capillaries and mural cells) using different combinations of fluorophores taking into consideration the abundance of the target antigens and the relative brightness of fluorophores.