Anti-GAT1 Antibody ValidAb^TM

HB7632 staining reveals the layer of GAT1 containing inhibitory interneurons in the cerebellum. Method: Brains were dissected from adult rats and fixed for 48hrs in 4% PFA before then incubated in 30% sucrose (in PBS) until the brains had sunk. A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 1% NaBH4 for 30 minutes then 0.05M glycine for 30 minutes. Sections were blocked in 2% BSA, 3% goat serum before incubation overnight in HB7632 (1:250, 1µg/ml) at 4°C. This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-rabbit DyLight 488 conjugated, Thermofisher 35552, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol . Images were captured using a Leica SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured using a 20x objective, 405nm (24.5% power, gain: 563V) and 488nm (30.1% power, gain: 742V) laser lines in a z-stack (1.63µm spacing). Deconvolution was carried out using Huygens Essential (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

HB7632 staining reveals the layer of GAT1 containing inhibitory interneurons in the cerebellum. Method: hippocampi were dissected from rat brains and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) for another 24hrs. A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 1% NaBH4 then 0.05M glycine for 30 minutes. Section were blocked in 1% BSA, 22.52mg/ml glycine before incubation overnight in HB7632 (1:250 dilution, 1µg/ml). This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-rabbit DyLight 594 conjugated, Thermofisher 35561, 1:500 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol . Images were captured using a Leica DMI6000B inverted epifluorescence microscope (20x objective) coupled to a Photometric-Prime95B monochrome digital camera with DAPI (5.6ms exposure) and TX2 (28.5ms exposure) filters. The image was processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool.

HB7632 revealed a band of approximately 65kDa corresponding to GAT1 only in the rat brain P2 fraction corresponding to GAT1’s membrane bound expression. Method: Rat brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (NEB Colour prestained , P7719S) before being run at 60V for 25 minutes followed by 130V for 80 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7632 at a 1:1,000 dilution (250ng/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (15g/l glycine, 1g/l SDS, 1% Tween 20, pH2.2) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).

HB7632 revealed a band of approximately 65kDa corresponding to GAT1 only in the mouse brain P2 fraction corresponding to GAT1’s membrane bound expression. Method: Mouse brain membrane (P2) and cytosol fractions were prepared following previous work (Molnar et al., 1993. Neuroscience 53:307-326) from freshly collected adult brains. Other tissue lysates were prepared following established protocols from freshly dissected tissue (see our guide on WB sample preparation). Samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (NEB Colour prestained , P7719S) before being run at 60V for 25 minutes followed by 130V for 80 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7632 at a 1:1,000 dilution (250ng/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (15g/l glycine, 1g/l SDS, 1% Tween 20, pH2.2) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 4 min exposure, 700nm channel: 30 sec exposure).

HB7632 worked well down to a 1:500 dilution (0.5µg/ml). Method: Brains were dissected from adult rats and fixed for 48hrs in 4% PFA before then incubated in 30% sucrose (in PBS) until the brains had sunk. A freezing microtome was used to cut 40µm transverse slices before sections were incubated in 1% NaBH4 for 30 minutes then 0.05M glycine for 30 minutes. Sections were blocked in 2% BSA, 3% goat serum for 2 hours before incubation overnight in HB7632 ranging in dilution from 1:250 to 1:2000 (1µg/ml to 0.125µg/ml) at 4°C. This was followed by a two hour incubation with secondary antibody (Polyclonal goat anti-rabbit DyLight 488 conjugated, Thermofisher 35552, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol . Images were captured using a Leica DMi8 inverted epifluorescence microscope (10x objective) in a z-stack (2.8µm spacing) coupled to a Leica DFC365FX monochrome digital camera with DAPI LP and FITC LP filters. Exposure times were as follows: 

• 1:250 – DAPI LP: 31.0ms @ 1x gain, FITC LP: 201.3ms @ 5.6x gain
• 1:500 – DAPI LP: 31.0ms @ 1x gain, FITC LP: 487.5ms @ 4.5x gain
• 1:1000 – DAPI LP: 31.0ms @ 1x gain, FITC LP: 487.5ms @ 7.8x gain
• 1:2000 – DAPI LP: 31.0ms @ 1x gain, FITC LP: 487.5ms @ 7.8x gain

Images were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (75px rolling ball radius) tool followed by Z-projection, stacking and montage creation. 

HB7632 produced consistent results down to a dilution of 1:2000 (125ng/ml). Method: P2 fractions were prepared from fresh mouse brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Mouse brain P2 fraction samples were loaded (20µg / lane) onto a 10% acrylamide gel alongside a protein ladder (BioRad Precision Plus Dual Colour, 1610374) before being run at 65V for 30 minutes followed by 180V for 60 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB7632. Each strip was incubated separately with a separate HB7632 concentration with this ranging from 0.5µg/ml (1:500 dilution) to 7.8ng/ml (1:32,000 dilution). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.