Hello Aamir.

Cyclin B1 is distributed throughout the cytoplasm even at G2/M transition when more cyclin B1 is translocated into the nucleus, but most of cyclin B are still in the cytoplasm and continuously degraded and synthesized.

Since you did not give any info about G2 arrest, I assume that G2 arrest should be verified by other probes. Some more intensive cells in the nuclei in the basal lamina are found.

Regards.

Did you include a negative control (secondary Ab alone)? A positive control (proliferating cells/tissues)? to make sure Ab is working as expected. May be the signal is because of autofluorescence or non-specific binding to a cytoplasmic protein.

Yes, I had a secondary only control. There was no signal. I did not do a positive control.

Did you get a positive result for any other nuclear protein using same protocol?

Yes, I used an alexa fluor 488 conjugated phh3 antibody that showed nuclear stain. I also used an alexa fluor 488 conjugated BrdU antibody that was nuclear as well.

Hello Aamir.

Cyclin B1 is distributed throughout the cytoplasm even at G2/M transition when more cyclin B1 is translocated into the nucleus, but most of cyclin B are still in the cytoplasm and continuously degraded and synthesized.

Since you did not give any info about G2 arrest, I assume that G2 arrest should be verified by other probes. Some more intensive cells in the nuclei in the basal lamina are found.

Regards.

Cyclin B1 is to a large extent cytoplasmic. You have some cells stained nuclear, BrdU is nuclear. It seems that you have the staining right. Protocol looks also good. You could still add Triton during the antibody incubation.

what is the nuclear protein you search in the nucleus by immunohistochemistry?. some proteins may exist in several cases simultaneously in the nucleus and in the cytoplasm.a large quantity of Cyclin B1 protein for example extent in the cytoplasm, also some glucocorticosteroid receptors. did you do a negative and positive control? tray them, and the washing time in a buffer solution must to be more long especially after the incubation with the second antibody

A negative control can help.

Try using a different buffer condition like Tris-EDTA, Ph 9

Try permeabilization for 5 more min

Cyclin B1 is also present in the cytoplasm.

Co staining with EDU, Ki67 or PCNA might help understand better.

Is the antibody specific to Mouse

Use 594 secondary

Amended on 31 August 2018

I have searched for Cyclin B1 in my human-protein database in vain (please see file; HepG2 Fucoidan). However, cell-cycle-related proteins are found in 3 biological specimens among 20 specimens. Fetal hepatocyte Hc has no cell-cycle-related proteins.

Cyclin-F isoform 1/G2/mitotic-specific cyclin-F has been detected at 4.5 μg/mg of cell protein in Cytoplasm or Nucleoplasm in hepatoma HepG2 (cultured without fucoidan). This cell has uniquely Primase/Helicase/primase complex protein (Varicella-Zoster virus/VSV/HHV-3) at 1.9 μg/mg of cell protein, and Replication protein E1 (Human papillomavirus type 60/HPV-60) at 0.9 μg/mg of cell protein, respectively.

Healed hepatocyte HepG2 (cultured with fucoidan) has Cell division control protein 2 homolog/Cyclin-dependent kinase 1/CDK1/Cell division protein kinase 1/p34 protein kinase at 0.26 μg/mg of cell protein, and has Minor capsid protein L2 (Human papillomavirus type 67/HPV-67) at 0.23 μg/mg of cell protein, respectively.

Serum of 4mo boy (with biotin deficiency and alopecia) has S phase cyclin A-associated protein in the ER/Zinc finger protein 291 at 12.6, and has Probable protein E5 (Human papillomavirus type 39/HPV-39) at 4.7 μg/mg of cell protein, respectively. Therefore, these cell-cycle-related proteins in HepG2 and serum of 4mo boy seem to be up-regulated by Varicella-Zoster virus/VSV/HHV-3 and Human papillomavirus/HPV.

Serum of GSD-1b patient (after liver transplantation at 2mo ago; biotin deficiency with alopecia has been cured by biotin therapy) has Cell cycle control protein 50C/CDC 50C/Transmembrane protein 30C at 13.9, Zinc finger protein 800 at 9.9, Membrane-spanning 4-domains subfamily A member 10 at 42.0, ATP synthase subunit gamma, mitochondrial at 3.1, and Serine/threonine-protein kinase PLK1/Centrosomal protein of 110 kDa/Polo-like kinase 1/Serine/threonine-protein kinase 13/Centriolar coiled-coil protein of 110 kDa isoform 1 at 2.1 μg/mg of serum protein, respectively (please see file; JMBT Alopecia). In this special case, gene control of host cell may be worked; i.e., Serine/threonine-protein kinase PLK1

is activated by growth-stimulating agents.

This special serum has Cysteine-rich motor neuron 2 protein (with Insulin-like growth factor (IGF)-activated receptor activity) at 5.1, and Epidermal growth factor receptor/EGF receptor at 3.6 μg/mg of serum protein, respectively.

Therefore, up-regulation of the cell-cycle-related proteins in this patient after liver transplantation seems to be occurred by IGF-1 and EGF.

By the way, two serum have expressed IGF-1 and EGF; i.e., serum of 12mo girl (with common cold) has Insulin-like growth factor 1A/IGF-I at 6.7, and serum of Gait disorder patient (32y, female) has Epidermal growth factor receptor 5/EGFR-5/Proto-oncogene c-ErbB-1 at 1.5 and Epidermal growth factor-like 2/Flamingo homolog 3 at 5.9 μg/mg of serum protein, respectively. These growth factors seems to be up-regulated by HHV-6. On the other hand, serum of GSD-1b patient (after liver transplantation at 2mo ago; biotin deficiency with alopecia has been cured by biotin therapy) has no HHV-6. Thus, induction of EGF and IGF-1 in patient of liver transplantation is not due to virus, but the stress of liver operation and nutrition level.

Human liver is a unique and famous organ to be able to regenerate. Therefore, it seems that the ability of regeneration in other human organs is strongly repressed. These findings are expected to deeply contribute in the Regenerative medicine.

Hi Aamir, may i ask you what did you conclude about this experiment?

i've been doing immunofluoresce with citrate buffer as antigen retrieval in spinal cord sections and i see the exact same signal. Mi negative controls do not have it but i'm still confuse because if a try another antibodies that are not necessary citoplasmatic, i see the same thing, so i was wondering if it is an artifact of citrate.

Thanks!