In-vivo distribution of NPs will be determined in all important organs 24 h after injection to allow for sufficient time for the onset of EPR effect. After 24 h, cancerous mice will be sacrificed for the collection of the tumor and organs. Samples will be harvested and immediately will be fixed in 4% paraform aldehyde, and then it will be dehydrated in a graded series of alcohol. After that it will be embed in paraffin. Slices 0.5 mm thick will be cut and will be stained with hematoxylin and eosin for microscopic examination. All sections will be photographed using a 400× light microscope. (Ren et al., 2012).

NPs concentration in different organ could be determined using other method and further data will be analyzed and will be discussed. Based on the hypothesis,NPs concentration in tumor site should be almost two times higher than normal tissue.

In my opinion, Inductively coupled plasma mass spectrometry or ICP Mass Spectrometry (ICP-MS), is the best way to quantify gold content in organs and you will obtain sufficient info to ensured your biodistribution profile and compare with others organs. By histology methods, using silver stain, you will obtain other kind of information, cell type biodistribution. For example, you will see kupffer cells full of gold in liver histology, right?

Good luck!!!

Thankyou so much Mr. Luciano Sobrevals

I quite agree with Mr. Luciano Sobrevals. One could also make use of TEM.

There are quantitative and qualitative method to determine biodistribution of Au nanoparticles in the tumor:

A-Quantitative

(Atomic Absorption Spectroscope (AAS) or Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) :

Weigh known amount of tumor tissue and digest it with nitric acid: perchloric acid (3:1) (30 ml: 10 ml) with heating and stirring at 200° c till the volume reaches 5 ml then complete with distilled water till 10 or 15 ml. Measure the gold with AAS or ICP-MS.

B-Qualitative (Confocal microscope or Transmission Electron Microscope)

Prepare section from the tissue using ultramicrotome to visualize AuNPs inside the tumor or follow procedures of sample preparation for confocal microscope (http://www.ncbi.nlm.nih.gov/pubmed/20949211)

Hope that useful

ICP-MS and AAS are among the best methods to quantify the amount of gold in tissues (kudos to Luciano and Omar). AAS is considered the gold standard. However, regarding the distribution of Au NPs in the tumor it's not so straightforward and simple. Finding the distribution inside the tumor is considered one of the serious challenges in the field and this an area of an active research. While one can get some information about it using histology, microscopy etc. the ultimate goal is visualize and quantify the distribution in vivo. Are we there yet? Not quite. Some techniques look very promising like photoacoustic tomography. Look at Lihong Wang publications, for example.

Another way you can determine the distribution of Au by IVIS imaging, if your Au particles are fluorescent/luminescent.

Easily you can check the biodistribution by ICP-MS or by IVIS imaging. Better you isolate the organs, digest in HNO3 and do ICP-MS to measure the amount of Au present in different organs.