What is the meaning of pale stained nuclei with hematoxylin?

I am staining pancreas with HE. I found beta cells show pale hematoxylin staining in conditional knockout mice compared to surrounding other islet cells, exocrine and acinar tissue " this means...

03 April 2018 234 1 View

Normalization by DNA content in glucose stimulated insulin secretion assay for islets?

I want to ask about normalization step: if I want to normalize by DNA content how I should isolate DNA after treatment of the islets with acidified ethanol?

07 August 2017 4,407 0 View

Has anybody faced difficulties with islets isolation from db/db mouse model?

Recently, I am trying to isolate islets from db/db mice. I use the same protocol I regularly use for islets isolation from mice but I can't get any islets or very few pale islets attached to...

01 February 2017 5,872 7 View

For murine islets, can I work on islets extracted from one mouse or I have to pool from several mice?

I am recently extracting murine islets. I see most researchers recommend pooling islets from several mice. I just want to check expression of my protein of interest in islets as an initial stage...

10 November 2016 9,330 3 View

What is the best internal control for western blots of mice lysates?

09 October 2016 6,228 3 View

Can I treat mice islets expressing tamoxifen induced cre in vitro with tamoxifen to knock out a floxed gene?

I have a mouse model that expresses modified Cre recombinase, so that only upon tamoxifen treatment of the animals they express Cre, thus KO of floxed gene can be induced. I want to isolate the...

07 August 2016 2,612 2 View

I am asking if somebody previously transfected islets of Langerhans isolated from mice with siRNA before. Is this going to work?

If this works what kind of tranfection reagent is recommended for the islets?

07 August 2016 4,448 9 View

Are beta actin and tubulin levels in islets affected by fasting ?

I am interested in knowing the expression of a certain protein in the islets under feeding and fasting conditions. I use the same total protein concentration for immunolotting. In my first trial I...

07 August 2016 4,264 1 View

I want to ask about measurement of insulin content in MIN6 cells?

I did a glucose stimulated insulin secretion assay for MIN6 cells. I measured the released insulin in the Kreb's Ringer buffer containing certain concentrations of glucose. In the end I collected...

06 July 2016 3,844 2 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 3 View

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

PUC19 as a transfer plasmid for lentiviral delivery?

Hello, Is it possible to use pUC19 as a transfer vector to be packed in using the second generation viral particles packaging system( pMD2.G; psPAX2 plasmids)? As far as I understand it there is...

28 February 2021 4,868 2 View

RIA. How to measure?

When you use RIA, with a control the unknown sample with antibodies, you bare in mind the binding sites of the antibodies. However you still need to measure labelled antigen (radioactive) and not...

28 February 2021 2,133 3 View

What is the most suitable resuspension buffer for TCA/acetone precipitated protein?

I need to extract protein from fermented carbon sources for Bradford assay. Most researchers experiencing insolubility of pellet in resuspension buffer. Please assist me to select most suitable...

26 February 2021 7,129 3 View

Does RNAiMAX transfection reagent work in serum free medium?

I diluted siRNA and RNAiMAX in opti-MEM and added to the cells which they were in the growth medium. Is it a right way? or should I culture cells in the opti-MEM medium for a while and not in...

26 February 2021 10,041 3 View

Did anyone ever try using pLVX-TetOne Plasmid with a copGFP as a selection marker instead of Puromycin?

When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...

25 February 2021 1,011 3 View