The most standardised dye to image A core plaques and CAA with multiphoton microscopy is Methoxy-X04 [13], which can be excited at 800?nm

The most standardised dye to image A core plaques and CAA with multiphoton microscopy is Methoxy-X04 [13], which can be excited at 800?nm. it cannot be detected with multiphoton microscopy. Representative of A cranial windows was implanted in APP/PS1 Tg mice and Wt littermates. Three weeks later, they were injected with HS-84 via retro-orbital and subjected to intravital multiphoton microscopy. B. Representative in vivo multiphoton microscopy images of HS-84 in Wt (top) and APP:PS1 Tg mouse (middle and bottom). Pictures show amyloid Rabbit polyclonal to HIRIP3 plaques and CAA labelled with HS-84 (green), Dextran Texas Red (red), Brivanib alaninate (BMS-582664) and merge of both channels. Scale bar represents 100?m and applies to all pictures. gene carrying the Swedish mutation K594n/M595?L and the exon 9 deletion mutation in the gene. In this model, amyloid pathology (A plaques and CAA) start to deposit around 5-months of age [7]. Six- Brivanib alaninate (BMS-582664) to 11-month-old APP:PS1 Tg mice of either sex, along with age-matched Wt littermate controls were used; 2) 8C11?month-old rTg4510 Tg mice of either sex (The Jackson laboratory, Tg (Camk2a-tTA)1Mmay Fgf14Tg (tetO-MAPT*P301L)4510Kh strain that expresses the tetracycline-controlled transactivator protein (tTA) [8]), along with age-matched Wt littermate controls were used, and 3) APP:PS1-rTg4510 (FVBB6F1rTg4510(App/PSEN1)85) (APP:PS1-rTg4510 mice), which exhibits mixed pathology of amyloid plaques and NFTs [9]. Mice were socially housed at 3C4 animals per cage with ad libitum access to food and water on a 12/12?h light/dark cycle with controlled conditions of temperature and humidity. Cranial windows implantation and LCO delivery Cranial windows medical procedures was performed as previously described [10] with minor modifications. Mice were anesthetized with 1.5% (vol/vol) isoflurane and placed in a stereotactic apparatus. A piece of skull (measuring 3?mm in diameter) over the left somatosensory Brivanib alaninate (BMS-582664) cortex was removed, replaced with a 5?mm diameter glass coverslip and fixed with a mixture of Krazy Glue and dental cement [11]. Body temperature was maintained at 37C throughout the full Brivanib alaninate (BMS-582664) procedure by using a heated pad. Mice were given buprenorphine (0.1?mg/kg) for 3?days following surgery, and were allowed to recover for at least 3 weeks before they were imaged. HS-84 and HS-169 were synthesized as described previously [3] and diluted with PBS to a stock concentration of 5?mg/mL. To label amyloid pathology and NFTs in vivo, 150?nmol of LCO in 150?l PBS [3] was intravenously delivered via retro-orbital injection [12]. A subset of APP:PS1 Tg mice were injected IP with Methoxy-X04 (363?nmol in 280?l PBS) 24?h before the imaging session to produce high contrast images of A plaques (emission 460C500?nm) [13]. To label NFTs in vivo with Thiazine Red in rTg4510 mice [14], dura matter was removed and 0.5?mg/ml Thiazine Red in PBS was topically applied onto the brain for 1?h, and then thoroughly washed with PBS. An 8?mm cranial windows was implanted and sealed with a mixture of Krazy Glue and dental cement. HS-84 was injected 1 week before the experiment. In vivo multiphoton imaging and data analysis In vivo imaging was performed on anesthetized mice (1.5% isoflurane). Images of amyloid pathology, NFTs and dextran angiograms were obtained using an Olympus FluoView FV1000MPE multiphoton Brivanib alaninate (BMS-582664) laser-scanning system mounted on an Olympus Bx61WI microscope and an Olympus 25x dipping objective (NA?=?1.05). A Deep-See Mai Tai Ti:Sapphire mode-locked laser (Mai Tai; Spectra-physics) generated two-photon excitation at 800?nm, and three photomultiplier tubes (PMTs) (Hamamatsu) collected emitted light in the range of 380C480, 500C540 and 560C650?nm [15]. Settings and laser power remained unchanged throughout the different imaging sessions. Either Texas Red dextran or fluorescein dextran (70,000?Da?MW; 12.5?mg/mL in PBS; Molecular Probes) was retro-orbitally injected before every imaging session to provide a fluorescent angiogram. The brain was imaged at depths of up to 200?m from.