Mice were transcardially perfused with cold phosphate-buffered saline (PBS, pH 7.4), followed by 4% paraformaldehyde (PFA) in PBS. Brains were removed from the skull and post-fixed in 4% PFA for 1–2 days at 4°C. Free-floating coronal sections (40 μm) were collected using a vibrating microtome (Thermo Scientific HM 650V) and washed twice for 10 minutes each in PBS containing 0.3% triton X-100. Sections were then incubated in blocking buffer (20% donkey serum in PBS) for 1 hour at room temperature, with gentle agitation. Incubation in primary antibody (anti-β-galactosidase, Invitrogen A-11132; 1∶200) was carried out in the same media at 4°C with gentle agitation, for 48 hours. After removal of primary antibody, sections were washed four times for 15 minutes each in PBS at room temperature. Sections were then incubated overnight at 4°C with a fluorescently tagged secondary antibody (AlexaFluor-488, 1:1000), diluted in PBS. Sections were washed briefly in PBS and mounted using DAPI Fluoromount-G (Southern Biotech, Birmingham, AL). Image acquisition and processing were performed at The Light Microscopy Facility at the Max Planck Florida Institute with a LSM 880 Zeiss confocal microscope controlled with ZEN 2011 software (Carl Zeiss, Oberkochen, Germany).
A 11132
Manufactured by Thermo Fisher Scientific
Sourced in Australia
The A-11132 is a high-precision laboratory instrument designed for use in a variety of scientific and research applications. It is capable of performing accurate measurements and analysis of various samples and materials. The core function of this product is to provide reliable and consistent data to support the research and development activities of our customers.
Automatically generated - may contain errors
Lab products found in correlation
Dapi fluoromount g, by Southern Biotech (2 mentions)
Zen 2011, by Zeiss (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Hm650v, by Thermo Fisher Scientific (2 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
A6455, by Thermo Fisher Scientific (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Na934, by GE Healthcare (1 mentions)
4 protocols using a 11132
1
Immunohistochemical Analysis of β-Galactosidase
2
Western Blot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western blotting, samples in SDS-PAGE loading buffer were separated by 8% or 12% gels prepared with WIDE RANGE Gel buffer (Nacalai Tasque) according to the manufacturer’s instructions, transferred onto a PVDF membrane and then subjected to immuno-detection using antibodies against GFP (A-6455; Invitrogen), LacZ (A-11132; Invitrogen), MifM34 (link), or c-Myc (A-14; Santa Cruz Biotechnology) as described previously34 (link). Images were obtained and analyzed using Amersham Imager 600 (GE Healthcare) luminoimager.
3
GPR37L1 Protein Detection and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue homogenates and crude membranes were prepared as previously described [15 (link), 16 (link)]. SDS-PAGE and immunoblotting was performed as previously described [15 (link)]. GPR37L1 was detected using goat anti-GPR37L1 (C-12) antibody (1:1000; sc-164532, Santa Cruz Biotechnology, USA) that was previously confirmed to be GPR37L1-specific using knockout tissue [15 (link)] and then rabbit anti-goat antibody (1:7500; 61-1620, Invitrogen). β-galactosidase was detected using rabbit anti-β-galactosidase antibody (1:5000; A-11132; Invitrogen) and then donkey anti-rabbit antibody (1:15000, NA934, GE Healthcare, Australia). GAPDH was detected using rabbit anti-GAPDH antibody (1:10000; 14C10, Cell Signaling, USA) and then donkey anti-rabbit antibody (1:20000, NA934, GE Healthcare). SuperSignal™ West Pico (34080, Invitrogen) or Clarity™ (1705060, Bio-Rad, USA) were used as chemiluminescent substrates. Densitometry was performed using ImageJ software (https://imagej.nih.gov/ij/ ) on non-saturated chemiluminescent exposures. Pixel density of background was measured and subtracted from subsequent measurements taken on the same image. Pixel density of GPR37L1 was measured in reference to the predominant, cleaved receptor species [15 (link)] and adjusted to equivalent protein abundance using each sample’s own GAPDH pixel density score.
4
Immunohistochemical Analysis of β-Galactosidase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were transcardially perfused with cold phosphate-buffered saline (PBS, pH 7.4), followed by 4% paraformaldehyde (PFA) in PBS. Brains were removed from the skull and post-fixed in 4% PFA for 1–2 days at 4°C. Free-floating coronal sections (40 μm) were collected using a vibrating microtome (Thermo Scientific HM 650V) and washed twice for 10 minutes each in PBS containing 0.3% triton X-100. Sections were then incubated in blocking buffer (20% donkey serum in PBS) for 1 hour at room temperature, with gentle agitation. Incubation in primary antibody (anti-β-galactosidase, Invitrogen A-11132; 1∶200) was carried out in the same media at 4°C with gentle agitation, for 48 hours. After removal of primary antibody, sections were washed four times for 15 minutes each in PBS at room temperature. Sections were then incubated overnight at 4°C with a fluorescently tagged secondary antibody (AlexaFluor-488, 1:1000), diluted in PBS. Sections were washed briefly in PBS and mounted using DAPI Fluoromount-G (Southern Biotech, Birmingham, AL). Image acquisition and processing were performed at The Light Microscopy Facility at the Max Planck Florida Institute with a LSM 880 Zeiss confocal microscope controlled with ZEN 2011 software (Carl Zeiss, Oberkochen, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!