rabbit anti 5ht 2a receptor  (Bioss)

Journal: Translational Psychiatry

Article Title: Intranasal delivery of shRNA to knockdown the 5HT-2A receptor enhances memory and alleviates anxiety

doi: 10.1038/s41398-024-02879-y

Figure Lengend Snippet: A – D Representative immunofluorescence images in human neurons following a 10-day treatment with scrambled AAV9 shRNA-AAV9 viral particles ( A , B ) or hu-HTR2A shRNA-AAV9 viral particles at MOI of 3 × 10 5 ( C , D ). Green fluorescence represents green fluorescence protein expression, while red fluorescence is indicative of 5HT-2A receptor protein following immunocytochemistry using an anti-mouse 5HT-2A receptor antibody at 1:50. Low level GFP expression, which served as a proxy for scrambled shRNA expression was observed in all cases (Fig. 1A). Panel B indicates robust expression of the 5HT-2A receptor protein in both cell bodies and neurites following treatment with the scrambled control. Panels C and D are representative images following treatment with hu-HTR2A shRNA-AAV9 and revealed stronger GFP expression ( C ). D Following hu-HTR2A shRNA-AAV9 treatment, a decrease in 5HT-2A fluorescent intensity was apparent. E ImageJ quantification of 5HT-2A immunofluorescence indicated a significant 27% decrease in hu-HTR2A shRNA-AAV9-treated neurons as compared to scrambled-treated (* p -value = 0.0019, N = 3 different samples for each condition). F Data show the results of qPCR real-time assays to analyze mRNA levels of Htr2a following extraction of RNA iPSC differentiated neurons in either vehicle-controls (blue bar) or scrambled treated (green bar), and hu-HTR2A shRNA (red bar). Neurons were treated on day 1 at a cell density of 200 K/well and cells pelleted and frozen on day 16. Results display the relative change in expression using GAPDH as an internal control. Real-time PCR results represent a total of 3 separate treatments for each condition in which cells were pooled and frozen at −80 °C. PCR experiments were performed in triplicate. The results indicated a significant 34% decrease in htr2a mRNA expression as compared to vehicle controls, where *denotes significant difference between the two groups, p = 0.012. Relative mRNA levels were not significantly different between vehicle control and scrambled-treated cells ( p -value = 0.502) or between scrambled- and shRNA-treated neurons ( p -value = 0.094).

Article Snippet: Briefly, all tissue sections were labeled with anti-GFP antibody (rabbit mAB #2956) 1:1000 (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-5HT-2A receptor antibody (rabbit polyclonal, #24288) at 1:500 dilution (Immunostar, Hudson, WI).

Techniques: Immunofluorescence, shRNA, Fluorescence, Expressing, Immunocytochemistry, Control, Extraction, Real-time Polymerase Chain Reaction

Journal: Translational Psychiatry

Article Title: Intranasal delivery of shRNA to knockdown the 5HT-2A receptor enhances memory and alleviates anxiety

doi: 10.1038/s41398-024-02879-y

Figure Lengend Snippet: Following behavioral analysis, a subset of brains from these mice were fixed, and 4 µm paraffin-embedded sagittal tissue sections were stained with a specific antibody against GFP (green fluorescence, 1:1000), anti-5HT2A receptor (red fluorescence, 1:500), or Hoechst nuclear labeling (blue fluorescence). Whole slide imaging was performed using a Pannoramic Midi II scanner (see “Materials and methods” for details). A Representative, low-field immunofluorescence sagittal image following treatment with MeCP2-GFP-mouse HTR2A-shRNA identified neuronal labeling in most brain regions including the olfactory bulb (OB), cortex, cerebellum, and numerous sub-cortical areas including the interpeduncular nucleus (IPN), a major connectome for stress-mediated pathways. Three different brain regions were examined at higher magnification including the cerebellum ( B – G ), IPN ( H – M ), and olfactory bulb ( N – S ). Data are representative images from either vehicle controls in each brain region ( B – D , H – J , or N – P ) and AAV-treated mice ( E – G , K – M , or Q – S ). Treatment with MeCP2-GFP-mouse HTR2A-shRNA led to a general pattern of less robust staining profile of the 5HT2A receptor in cell body regions and apical dendrites (merged image Panel G as an example). All scale bars represent 50 µm. T – V Quantitative analysis using ImageJ software indicated a significant decrease in 5HT-2A receptor fluorescence intensity of AAV-treated mice (red bar) versus vehicle-controls (green bar) in both total brain sections ( T ) and olfactory bulb ( U ). Although there was a trend for a decrease in 5HT-2A receptor fluorescence intensity in the cerebellum, data did not reach significance. Data represent the mean gray value ± SD in three different cases. *Denotes significant difference, p -value < 0.05. W , X Data show the results of qPCR real-time assays to analyze mRNA levels of Htr2a following extraction of total brain RNA from frozen brain ( W ) or olfactory tissue ( X ) in either vehicle-controls (black bars) or shRNA treated (red bars). Results display relative mRNA levels after 8-weeks of treatment with AAV9-MeCP2-GFP-mouse HTR2A as compared to vehicle-controls. Real-time PCR results represent a total of N = 5 animals for each group performed in triplicate ±SEM. *Denotes significant difference between the two groups, p = 0.006 in olfactory mRNA.

Article Snippet: Briefly, all tissue sections were labeled with anti-GFP antibody (rabbit mAB #2956) 1:1000 (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-5HT-2A receptor antibody (rabbit polyclonal, #24288) at 1:500 dilution (Immunostar, Hudson, WI).

Techniques: Staining, Fluorescence, Labeling, Imaging, Immunofluorescence, shRNA, Software, Extraction, Real-time Polymerase Chain Reaction

Journal: Translational Psychiatry

Article Title: Intranasal delivery of shRNA to knockdown the 5HT-2A receptor enhances memory and alleviates anxiety

doi: 10.1038/s41398-024-02879-y

Figure Lengend Snippet: A Aged male C57Bl6 mice were treated intranasally with vehicle or with 2.0 × 10 11 viral particles on day 1, and 5-weeks later tested behaviorally using a spontaneously alteration memory test. Mice treated with AAV9-CRISPR/Cas9 showed a significant increase in the percent spontaneous alterations ( p -value = 0.0007, N = 15 mice per group, asterisk, blue bar). B Representative, merged immunofluorescence image of vehicle-control animals depicting the presence of 5HT-2A receptor protein labeling in apical dendrites in the CA2/CA3 region of the hippocampus. The blue staining reflects nuclear staining with DAPI. As expected, there was no expression of GFP in vehicle controls. C Identical to Panel B with the exception that the merged image is from a AAV9-CRISPR/Cas9-treated mouse brain. In this case, strong GFP labeling was observed in cell bodies while there was an observed decrease in 5HT-2A receptor fluorescence. Images are representative of 3 separate mice for each group.

Article Snippet: Briefly, all tissue sections were labeled with anti-GFP antibody (rabbit mAB #2956) 1:1000 (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-5HT-2A receptor antibody (rabbit polyclonal, #24288) at 1:500 dilution (Immunostar, Hudson, WI).

Techniques: CRISPR, Immunofluorescence, Control, Labeling, Staining, Expressing, Fluorescence

Journal: Translational Psychiatry

Article Title: Intranasal delivery of shRNA to knockdown the 5HT-2A receptor enhances memory and alleviates anxiety

doi: 10.1038/s41398-024-02879-y

Figure Lengend Snippet: The target sequence used to synthesize the shRNA is 100% conserved between mice and rats. To test whether shRNA-knockdown of the rat 5HT-2A receptor improves memory, Wistar rats (12 animals per group) were randomly assigned to two different groups consisting of vehicle- or AAV9-MeCP2-GFP-mHTR2A-shRNA. Following treatment on day 1, animals were assessed behaviorally 3- ( A – D ) and 5-weeks later ( E – H ). Details of the novel object recognition test can be found in the methods. At 3 weeks, there was no significance difference in the time spent or the number of physical contacts with the novel object during the acquisition (training) period ( p > 0.05) ( A , B ). Rats were tested 24-h later, and AAV9-treated rats (blue bars) showed a significant increase in both the contact-recognition index ( p < 0.000003) ( C ) and the time recognition-index ( p < 0.0003) ( D ). The same groups of rats were retested at 5-weeks and again no significant difference was noted in the time spent or the number of physical contacts with the novel object during the acquisition (training) period ( p > 0.05) ( E , F ). Twenty-four hours later there was a significant increase in the contact-recognition index ( p < 0.01) ( G ), however, there was no significant difference in the contact-recognition index ( p = 0.114) ( H ). I Data show the results of qPCR real-time assays to analyze mRNA levels of Htr2a following extraction of rat brain RNA. Results display relative mRNA levels after 5-weeks post-treatment with AAV9-HTR2A-shRNA. Real-time PCR results represent a total of N = 5 animals for each group performed in triplicate ±SEM. *Denotes significant difference between the two groups, p = 0.038. J Representative, merged immunofluorescence image of vehicle-control animals depicting the presence of 5HT-2A receptor protein labeling (red fluorescence) within the olfactory bulb. K Representative, merged immunofluorescence image of AAV9-HTR2A-shRNA-treated animals. The blue staining reflects nuclear staining with DAPI. As expected, there was no expression of GFP in vehicle controls ( J , top panel) while strong GFP labeling was observed in cell bodies of neurons of shRNA-treated rats ( K , bottom panel). Images are representative of 3 separate mice for each group.

Article Snippet: Briefly, all tissue sections were labeled with anti-GFP antibody (rabbit mAB #2956) 1:1000 (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-5HT-2A receptor antibody (rabbit polyclonal, #24288) at 1:500 dilution (Immunostar, Hudson, WI).

Techniques: Sequencing, shRNA, Knockdown, Extraction, Real-time Polymerase Chain Reaction, Immunofluorescence, Control, Labeling, Fluorescence, Staining, Expressing