Animal Injections and Tissue Processing: All animals were treated according to the NIH Guidelines for Animal Care and Use as approved by the Caltech Institutional Animal Care and Use Committee. Three female BALB/c mice (Jackson Laboratory) were each injected with 4.5×1011 nanoparticles via lateral tail vein for all nanoparticle formulations. The mice were euthanized by CO2 asphyxiation after 12 hrs. The brains were resected and bisected with a manual mid-sagittal cut before fixing both hemispheres overnight in 10% neutral buffered formalin (Sigma). Individual hemispheres were dehydrated in increasing concentrations of ethanol (3 x 30 min each for 50, 70, 95 and 100% EtOH), followed by xylenes (3 x 30 min) and 50:50 xylene:paraffin mixture (1 x 30 min). The tissues were then incubated in molten paraffin (3 x 1 hr) at 60°C. The brains were placed in a paraffin mold and stored at 4°C until sectioning. A Leica 1512 microtome was used to cut 5μm sections.
Silver Enhancement Staining of Mouse Brains: All glassware used was washed with Farmer’s Solution (9 parts 10% sodium thiosulfate and 1 part 10% potassium ferricyanide) for 20 min prior to tissue processing to reduce non-specific silver staining. Paraffin-embedded sections were deparaffinized in xylenes, rehydrated using decreasing concentrations of ethanol and washed in pure water (3 x 1min). Silver enhancement solution (Ted Pella) was added to the sections for 20 min at RT. The stained sections were immediately placed in pure water for 5 min followed by counterstaining with haematoxylin for 2 min at RT. They were then dehydrated with increasing concentration of ethanol and xylenes and mounted with Permount (Fisher).
Sorry to hear you’re having difficulty with the stain. I’ve attached the protocol I used to perform the stains in the manuscript you cited.
One major difference between that work and yours is the size of nanoparticle. We worked solely with 50nm gold cores in that paper, which show clear, individual NP’s at low magnification when stained under the right conditions. We used 5nm in one of our earlier manuscripts (Wiley et al here: http://www.pnas.org/content/110/21/8662.full. These are listed as 20nm particles, which are 5nm gold cores with a surface PEG-Tf coating), and individual NP’s were much harder to visualize (see Fig S5).
Using smaller NP’s may necessitate modifying the staining conditions to improve signal, most importantly the length of time the tissue is incubated with the silver solution. Our protocols are also based off 5um tissue sections as thicker sections may take longer to develop. We recommend using the Ted Pella silver enhancement kit for staining too as we found other brands’ kits to provide inconsistent results. A final tip is to perform your stains simultaneously with both a positive (tissue spiked with dilute NP’s) and negative control (tissue with no NP’s) to verify your stain is working and what signal you see is due to NP’s.
Hope this helps. Good luck with your experiments,