26 Questions 16 Answers 0 Followers
Questions related from David J Walker
Hi there, I'm rather ashamed to ask this question but need re-assurance and advice for making dilutions of my primers for a 5'RACE PCR. I am trying to amplify heavy and light chain antibody...
01 May 2024 7,976 0 View
Hi there, I'm isolating cells from spleen tissue for flow cytometry. Last time from a single spleen I got around 40 million cells total but I reckon I can get much more with some refinement....
24 May 2023 7,064 2 View
I'm planning to make conditioned media from various cell lines for some experiments and have come across various protocols, which all differ in their methods. My current plan is: 1) Culture...
12 January 2023 8,415 2 View
Hi there, I wish to run analysis of counts from a published RNAseq experiment to look for genes that are differentially expressed in a knockdown/knockout cell line, with the aim to identify...
09 October 2022 3,688 0 View
I've been piloting clonogenic survival assays to look at long-term effects of of drugs in colon cancer cell lines (SW620, SW480, HT-29). My initial pilot assays in 12-well plates were successful...
18 March 2022 7,636 1 View
I've been trying for several weeks to get consistent and successful transient transfection of glosensor DNA (promega) into Sw620 and Sw480 colon cancer cells in order to measure cAMP levels by...
23 November 2021 8,240 3 View
Hi there, I've developed an algorithm in FIJI for quantifying Iba+ cells via DAB counterstain on IHC brain sections. My algorithm does a pretty good job of identifying the soma which allows for...
29 July 2020 3,494 3 View
I've been working with murine neural stem cells as a 2d monolayer which is achieved by adding laminin (ECM) directly to the culture media, without the need to coat the TC plastics. However, I've...
05 February 2020 8,352 2 View
I have multiple sig pairwise comparisons generated from a 3-way interaction from a mixed model GLM. I want to present these comparisons in my thesis but wish to do it concisely so it is easy to...
15 June 2018 8,121 3 View
Hi there, I recently lysed some tissue samples but found my lysis buffer interfered with the bca protein assay so my protein concs were not accurate. The buffer I used (0.01M PBS pH 7.4, 20%...
17 December 2017 1,359 3 View
I'm planning on looking at oxidative stress markers in quail red blood cells we have stored at -80. I'm planning to run them on an ELISA measuring protein carbonyls and SOD concentrations among...
31 July 2017 432 3 View
Hi there, I need assistance with interpreting the outcome of my pairwise comparisons from my datasets. I've been running a glmer mixed models and selecting the best model using the AIC criteria....
28 July 2017 273 2 View
I have a lot of tissue sections with various treatments and for my control sections, I've determined the optimal exposure time for each fluorophore. These are fine for the DAPI and 488 channels...
04 February 2017 4,540 3 View
Hi, I have several dual-labelled fluorescent images of brain tissue where I have obvious colocalisation between my protein of interest (BDNF, imaged at Alexafluor 594) and the nuclei (DAPI...
18 December 2016 9,344 2 View
Hi there, Yet another IF-related question. I'm going to be dual-labelling free-floating tissue sections with fluorescent secondary abs. After the final wash steps, I will be mounting sections...
23 June 2016 7,840 3 View
Hi there, I have two targets I want to dual label for immunofluorescence. One ab is raised in sheep and other in rabbit. So I need an anti-sheep and anti-rabbit secondary alexafluor. Now I...
20 June 2016 3,405 9 View
Hi there, I'm planning some immunofluorescence where I want to dual label either BDNF or TRKB with caspase-3. However, for the species of tissue I'm using, I need to use an antibody that hasn't...
29 May 2016 4,373 2 View
Hi all, I need some advice for mounting brain tissue sections (50um) for immunofluorescenct detection. At the moment I plan to dual stain with two different primary antibodies and then mount in...
17 May 2016 3,708 3 View
I require a mRNA marker for microlgia for qPCR analysis and was wondering if IFNg would be a suitable candidate. I am looking at microglia in quail and commerical gene sequences are hard to come...
07 July 2015 6,590 3 View
I've come across a neat protocol for measuring immune response in plasma which uses bioluminescence. However, I was wondering if it was possible if the same samples can then be processed for...
04 March 2015 10,000 1 View
I'm looking for an IHC or mRNA marker that specifically identifies ramified (unactivated) microglia so I can distinguish between an inactive and active phenotype. Can anyone help please? Thanks
29 January 2015 9,899 8 View
I am preparing ACSF for organotypic slice culture experiments (which I haven't done before) but I want to make a 10x stock and I want to make sure I have separated the correct components as some...
08 January 2015 2,663 0 View
My question has two parts re the organotypic slice culture technique: How long can tissue (in this case brain) remain embedded in agarose prior to slicing on vibratome (4% low-melt agarose in...
27 December 2014 6,312 1 View
I am planning to perform organotypic slice culture experiments and I was initially planning to perform a TUNEL assay to test if the neurons in my cultures will survive. However, I was wondering...
05 December 2014 9,085 2 View
Hi there, I want to generate NADPH oxidase (NOX)-specific induction of ROS in vitro and was wanting to try and avoid using H2O2 since this is a downstream product and ROS is naturally generated in...
29 October 2014 446 4 View
I am looking for papers detailing a method to isolate primary neurons from an avian species and establish them in culture. In particular, I would like an idea of what sort of cytokines/growth...
13 March 2014 8,640 2 View
